Acylated glp-1/glp-2 dual agonists

ABSTRACT

A compound having agonist activity at the GLP-1 (glucagon-like-peptide 1) and GLP-2 (glucagon-like peptide 2) receptors, and a pharmaceutical composition containing the compound or a pharmaceutically acceptable salt or solvate thereof in admixture with a pharmaceutically acceptable carrier, an excipient or a vehicle are provided. The compound can be used, inter alia, in the prophylaxis or treatment of intestinal damage and dysfunction, regulation of body weight, and prophylaxis or treatment of metabolic dysfunction.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

Any and all applications for which a foreign or domestic priority claimis identified in the Application Data Sheet as filed with the presentapplication are hereby incorporated by reference under 37 CFR 1.57.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 19, 2019 isnamed “50412-110004_Sequence_Listing_8.19.2019_ST25” and is 262,589bytes in size.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to acylated compounds having agonistactivity at the GLP-1 (glucagon-like-peptide 1) and GLP-2 (glucagon-likepeptide 2) receptors. The compounds find use, inter alia, in theprophylaxis or treatment of intestinal damage and dysfunction,regulation of body weight, and prophylaxis or treatment of metabolicdysfunction.

Description of Related Art

Intestinal tissue is responsible for the production of both humanglucagon-like peptide 1 (GLP-1(7-36)) and human glucagon-like peptide 2(GLP-2 (1-33)) as they are produced by the same cells. Human GLP-2 is a33-amino-acid peptide with the following sequence:Hy-His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-OH(SEQ ID NO: 1). It is derived from specific posttranslational processingof proglucagon in the enteroendocrine L cells of the intestine and inspecific regions of the brainstem. GLP-2 binds to a singleG-protein-coupled receptor belonging to the class II glucagon secretinfamily. GLP-2 is co-secreted with GLP-1, oxyntomodulin and glicentin, inresponse to nutrient ingestion. Human GLP-1 is produced as a 30-aminoacid peptide with the following sequence:Hy-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-NH2(SEQ ID NO: 2).

GLP-2 has been reported to induce significant growth of the smallintestinal mucosal epithelium via the stimulation of stem cellproliferation in the crypts, and by inhibition of apoptosis in the villi(Drucker et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7911-7916). GLP-2also has growth effects on the colon. Furthermore, GLP-2 inhibitsgastric emptying and gastric acid secretion (Wojdemann et al., 1999, J.Clin. Endocrinol. Metab. 84: 2513-2517), enhances intestinal barrierfunction (Benjamin et al., 2000, Gut 47: 112-119), stimulates intestinalhexose transport via the upregulation of glucose transporters(Cheeseman, 1997, Am. J. Physiol. R1965-71), and increases intestinalblood flow (Guan et al., 2003, Gastroenterology, 125: 136-147).

GLP-1 has been described as a physiological incretin hormone and hasthus been mostly reported to augment an insulin response after an oralintake of glucose or fat. It is, however, generally understood thatGLP-1 lowers glucagon concentrations, has beneficial effects oninhibition of fast bowel movements (Tolessa et al., 1998, Dig. Dis. Sci.43(10): 2284-90), and slows gastric emptying.

WO2013/164484 discloses GLP-2 analogues which comprise one or moresubstitutions compared to h[Gly2]GLP-2 and which may have the propertyof an altered GLP-1 activity, and their medical use.

WO2016/066818 describes peptides having dual agonist activity at theGLP-1 and GLP-2 receptors, and proposes medical uses thereof. However,there remains a need for further compounds which combine effectiveagonist activities at both receptors with acceptable levels ofstability.

SUMMARY OF THE INVENTION

Broadly, the present invention relates to compounds which have agonistactivity at the GLP-1 (glucagon-like peptide 1) and GLP-2 (glucagon-likepeptide 2) receptors, e.g. as assessed in in vitro potency assays. Suchcompounds are referred to in this specification as “GLP-1/GLP-2 dualagonists,” or simply “dual agonists.” Thus, the compounds of the presentinvention have activities of both GLP-1 (7-36) and GLP-2 (1-33).

In a first aspect there is provided a GLP-1/GLP-2 dual agonistrepresented by the formula:

R¹-X*-U-R²

wherein:

R¹ is hydrogen (Hy), C₁₋₄ alkyl (e.g., methyl), acetyl, formyl, benzoylor trifluoroacetyl;

R² is NH₂ or OH;

X* is a peptide of formula I:

(I) (SEQ ID NO: 3) H-X2-EG-X5-F-X7-X8-E-X10-X11-TIL-X15-X16-X17-A-X19-X20-X21-FI-X24-WL-X27-X28-X29-KIT-X33

wherein:

X2 is Aib or G;

X5 is T or S;

X7 is T or S;

X8 is S, E or D;

X10 is L, M, V or ψ;

X11 is A, N or S;

X15 is D or E;

X16 is G, E, A or ψ;

X17 is Q, E, K, L or ψ;

X19 is A, V or S;

X20 is R, K or ψ;

X21 is D, L or E;

X24 is A, N or S;

X27 is I, Q, K, H or Y;

X28 is Q, E, A, H, Y, L, K, R or S;

X29 is H, Y, K or Q;

X33 is D or E;

U is absent or a sequence of 1-15 residues each independently selectedfrom K, k, E, A, T, I, L and ψ;

the molecule contains one and only one Ψ, wherein Ψ is a residue of K,k, R, Orn, Dap or Dab in which the side chain is conjugated to asubstituent having the formula Z¹— or Z¹—Z²—, wherein

Z¹— is CH₃—(CH₂)₁₀₋₂₂—(CO)— or HOOC—(CH₂)₁₀₋₂₂—(CO)—; and

—Z²— is selected from —Z^(S1)—, —Z^(S1)—Z^(S2)—, —Z^(S2)—Z^(S1),—Z^(S2)—, —Z^(S3)—, —Z^(S1)Z^(S3)—, —Z^(S2)Z^(S3)—, —Z^(S3)Z^(S1)—,—Z^(S3)Z^(S2)—, —Z^(S1)Z^(S2)Z^(S3)—, —Z^(S1)Z^(S3)Z^(S2)—,—Z^(S2)Z^(S1)Z^(S3)—, —Z^(S2)Z^(S3)Z^(S1)—, —Z^(S3)Z^(S1)Z^(S2)—,—Z^(S3)Z^(S2)Z^(S1)—, —Z^(S2)Z^(S3)Z^(S2)— wherein

Z^(S1) is isoGlu, β-Ala, isoLys, or 4-aminobutanoyl;

Z^(S2) is -(Peg3)_(m)- where m is 1, 2, or 3; and

—Z^(S3)— is a peptide sequence of 1-6 amino acid units independentlyselected from the group consisting of A, L, S, T, Y, Q, D, E, K, k, R,H, F and G;

and wherein at least one of X5 and X7 is T;

or a pharmaceutically acceptable salt or solvate thereof.

The various amino acid positions in peptide X* of the formulae providedhere are numbered according to their linear position from N- toC-terminus in the amino acid chain.

In the present context, β-Ala and 3-Aminopropanoyl are usedinterchangeably.

Dual agonists having aspartic acid (Asp, D) at position 3 and glycine(Gly) in position 4 can be very potent agonists at the GLP-1 and GLP-2receptors. However, this combination of substitutions results incompounds which are unstable and may not be suitable for long termstorage in aqueous solution.

Without wishing to be bound by theory, it is believed that the Asp atposition 3 may isomerise to iso-Asp via a cyclic intermediate formedbetween the carboxylic acid functional group of its side chain and thebackbone nitrogen atom of the residue at position 4.

It has now been found that molecules having glutamic acid (Glu, E) atposition 3 instead of Asp are much less susceptible to such reactionsand hence may be considerably more stable when stored in aqueoussolution. However, replacement of Asp with Glu at position 3 inmolecules having a lipophilic substituent in the middle portion of thepeptide (e.g., at or near to positions 16 and 17) tends to reduce thepotency at one or both of the GLP-2 receptor and the GLP-1 receptor,even though Glu is present at position 3 of the native GLP-1 molecule.Simultaneously incorporating a Thr residue at one or both of positions 5and 7 appears to compensate for some or all of the lost potency. It isbelieved that further improvements in potency are also provided byincorporation of His (H), Tyr (Y), Lys (K) or Gln (Q) at position 29instead of the Gly (G) and Thr (T) residues present in wild type humanGLP-1 and 2 respectively.

In some embodiments of formula I:

X2 is Aib or G;

X5 is T or S;

X7 is T or S;

X8 is S;

X10 is L or ψ;

X11 is A or S;

X15 is D or E;

X16 is G, E, A or ψ;

X17 is Q, E, K, L or ψ;

X19 is A or S;

X20 is R or ψ;

X21 is D, L or E;

X24 is A;

X27 is I, Q, K, or Y;

X28 is Q, E, A, H, Y, L, K, R or S;

X29 is H, Y or Q; and

X33 is D or E.

Where ψ is not at X16 or X17, it may be desirable that X16 is E and X17is Q.

In some embodiments, X11 is A and X15 is D. In other embodiments, X11 isS and X15 is E. In further embodiments, X11 is A and X15 is E.

In some embodiments, X27 is I.

In some embodiments, X29 is H. In certain of these embodiments, X28 is Aand X29 is H, or X28 is E and X29 is H.

In some embodiments, X29 is Q and optionally X27 is Q.

In some embodiments, the residues at X27-X29 have a sequence selectedfrom:

IQH;

IEHF

IAH;

IHH;

IYH;

ILH;

IKH;

IRH;

ISH;

QQH;

YQH;

KQH;

IQQ;

IQY;

IQT; and

IAY.

In some embodiments, X* is a peptide of formula II:

(II) (SEQ ID NO: 4) H-X2-EG-X5-F-X7-SELATILD-X16-X17-AAR-X21-FIAWLI-X28-X29-KITD

wherein:

X2 is Aib or G;

X5 is T or S;

X7 is T or S;

X16 is G or ψ;

X17 is Q, E, K, L or ψ;

X21 is D or L;

X28 is Q, E, A, H, Y, L, K, R or S;

X29 is H, Y or Q;

In some embodiments of Formula I or Formula II, X16 is ψ and X17 is Q,E, K or L. For example, X17 may be Q, or X17 may be selected from E, Kand L. In other embodiments, X16 is G and X17 is ψ.

It may be desirable that X21 is D.

X28 may be selected from Q, E and A, e.g. it may be Q or E. In someresidue combinations, Q may be preferred. In others, E may be preferred,including but not limited to when X16 is G and X17 is ψ.

Alternatively, X28 may be selected from A, H, Y, L, K, R and S.

X* may be a peptide of formula III:

(III) (SEQ ID NO: 5) H[Aib]EG-X5-F-X7-SE-X10-ATILD-X16-X17-AA-X20-X21-FIAWLI-X28-X29-KITD

wherein:

X5 is T or S;

X7 is T or S;

X10 is L or ψ;

X16 is G, E, A or ψ;

X17 is Q, E, K, L or ψ;

X20 is R or ψ;

X21 is D or L;

X28 is E, A or Q;

X29 is H, Y or Q;

and at least one of X5 and X7 is T.

X* may be a peptide of formula IV:

(IV) (SEQ ID NO: 6) H[Aib]EG-X5-F-X7-SELATILD-X16-X17-AAR-X21-FIAWLI-X28-X29-KITD

wherein:

X5 is T or S;

X7 is T or S;

X16 is G or ψ;

X17 is E, K, L or ψ;

X21 is D or L;

X28 is E or A;

X29 is H, Y or Q;

and at least one of X5 and X7 is T.

In some embodiments of any of formulae I to IV, X16 is ψ and X17 is E, Kor L.

In other embodiments of formula I to IV, X16 is G and X17 is IP.

In either case, the following combinations of residues may also beincluded:

X21 is D and X28 is E;

X21 is D and X28 is A;

X21 is L and X28 is E; or

X21 is L and X28 is A.

X* may be a peptide of formula V:

(V) (SEQ ID NO: 7) H[Aib]EG-X5-F-X7-SELATILD-Ψ-QAARDFIAWLI-X28-X29- KITD

wherein

X5 is T or S;

X7 is T or S;

X28 is Q, E, A, H, Y, L, K, R or S, e.g., Q, E, A, H, Y or L;

X29 is H, Y or Q;

and at least one of X5 and X7 is T.

In some embodiments of formula III, X28 is Q or E. In some embodimentsof formula III, X28 is Q. In other embodiments, X28 is A, H, Y, L, K, Ror S, e.g. A, H, Y or L.

In any of the formulae or embodiments described above, the dual agonistcontains one of the following combinations of residues:

X5 is S and X7 is T;

X5 is T and X7 is S;

X5 is T and X7 is T.

It may be preferred that X5 is S and X7 is T, or X5 is T and X7 is T.

In any of the formulae or embodiments described above, it may bedesirable that X29 is H.

In some embodiments, ψ is a Lys residue whose side chain is conjugatedto the substituent Z¹— or Z¹—Z²—.

In some embodiments, Z¹—, alone or in combination with —Z²—, isdodecanoyl, tetradecanoyl, hexadecanoyl, octadecanoyl or eicosanoyl.

In some embodiments, Z¹—, alone or in combination with —Z²—, is:

13-carboxytridecanoyl, i.e., HOOC—(CH₂)₁₂—(CO)—;

15-carboxypentadecanoyl, i.e., HOOC—(CH₂)₁₄—(CO)—;

17-carboxyheptadecanoyl, i.e., HOOC—(CH₂)₁₆—(CO)—;

19-carboxynonadecanoyl, i.e., HOOC—(CH₂)₁₈—(CO)—; or

21-carboxyheneicosanoyl, i.e., HOOC—(CH₂)₂₀—(CO)—.

In some embodiments Z² is absent.

In some embodiments, Z² comprises Z^(S1) alone or in combination withZ^(S2) and/or Z^(S3).

In such embodiments:

—Z^(S1)— is isoGlu, β-Ala, isoLys, or 4-aminobutanoyl;

—Z^(S2)—, when present, is -(Peg3)_(m)- where m is 1, 2, or 3; and

—Z^(S3)— is a peptide sequence of 1-6 amino acid units independentlyselected from the group consisting of A, L, S, T, Y, Q, D, E, K, k, R,H, F and G, such as the peptide sequence KEK.

Z² may have the formula —Z^(S1)—Z^(S3)—Z^(S2)—, where Z^(S1) is bondedto Z¹ and Z^(S2) is bonded to the side chain of the amino acid componentof ψ.

Thus, in some embodiments, —Z²— is:

isoGlu(Peg3)₀₋₃;

β-Ala(Peg3)₀₋₃;

isoLys(Peg3)₀₋₃; or

4-aminobutanoyl(Peg3)₀₋₃.

In further embodiments, —Z²— is:

isoGlu-KEK-(Peg3)₀₋₃.

Specific examples of the substituent Z¹—Z²— are set out below. In someembodiments, Z¹—Z²— is [17-carboxy-heptadecanoyl]-isoGlu. For example, ψmay be K([17-carboxy-heptadecanoyl]-isoGlu). In some embodiments, Z¹—Z²—is:

[17-Carboxy-heptadecanoyl]-isoGlu-KEK-Peg3- (SEQ ID NO: 413);

[17-carboxy-heptadecanoyl]-isoGlu-Peg3-;

[19-Carboxy-nonadecanoyl]-isoGlu-;

[19-Carboxy-nonadecanoyl]-isoGlu-KEK-;

[19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3- (SEQ ID NO: 414);

[19-carboxy-nonadecanoyl]-isoGlu-KEK-Peg3-Peg3- (SEQ ID NO: 434);

[19-carboxy-nonadecanoyl]-isoGlu-Peg3-Peg3-;

[19-carboxy-nonadecanoyl]-isoLys-Peg3-Peg3-Peg3-;

[Hexadecanoyl]-βAla-;

[Hexadecanoyl]-isoGlu-; or

Octadecanoyl-.

For example, ψ may be:

K([17-Carboxy-heptadecanoyl]-isoGlu-KEK-Peg3);

K([17-carboxy-heptadecanoyl]-isoGlu-Peg3);

K([19-Carboxy-nonadecanoyl]-isoGlu);

K([19-Carboxy-nonadecanoyl]-isoGlu-KEK);

K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3);

K([19-carboxy-nonadecanoyl]-isoGlu-KEK-Peg3-Peg3);

K([19-carboxy-nonadecanoyl]-isoGlu-Peg3-Peg3);

K([19-carboxy-nonadecanoyl]-isoLys-Peg3-Peg3-Peg3);

K([Hexadecanoyl]-βAla-;

K([Hexadecanoyl]-isoGlu); or

K(Octadecanoyl).

When present, U represents a peptide sequence of 1-15 residues eachindependently selected from K (i.e., L-lysine), k (i.e., D-lysine) E(Glu), A (Ala), T (Thr), I (Ile), L (Leu) and P. For example, U may be1-10 amino acids in length, 1-7 amino acids in length, 3-7 amino acidsin length, 1-6 amino acids in length, or 3-6 amino acids in length.

Typically, U includes at least one charged amino acid (K, k or E) andpreferably two or more charged amino acids. In some embodiments itincludes at least 2 positively charged amino acids (K or k), or at least1 positively charged amino acid (K or k) and at least one negativelycharged amino acid (E). In some embodiments, all amino acid residues ofU (except for ψ, if present) are charged. For example, U may be a chainof alternately positively and negatively charged amino acids.

In certain embodiments, U comprises residues selected only from K, k, Eand ψ.

In certain embodiments, U comprises residues selected only from K, k,and ψ.

When U comprises only lysine residues (whether K or k), all residues mayhave an L-configuration or all may have a D-configuration. Examplesinclude K₁₋₁₅, K₁₋₁₀ and K₁₋₇, e.g., K₃, K₄, K₅, K₆ and K₇, especiallyK₅ and K₆. Further examples include k₁₋₁₅, k₁₋₁₀ and k₁₋₇, e.g. k₃, k₄,k₅, k₆ and k₇, especially k₅ and k₆.

Further examples of peptide sequences U include KEK (SEQ ID NO: 8),EKEKEK (SEQ ID NO: 9), EkEkEk (SEQ ID NO: 11), AKAAEK (SEQ ID NO: 12),AKEKEK (SEQ ID NO: 13) and ATILEK (SEQ ID NO: 14).

In any case, one of those residues may be exchanged for ψ. Where thesequence U contains a residue ψ, it may be desirable that the C-terminalresidue of U is ψ. Thus, further examples of sequences U includeK₁₋₁₄-ψ, K₁₋₉-ψ and K₁₋₆-ψ, e.g., K₂-ψ, K₃-ψ, K₄-ψ, K₅-ψ and K₆-ψ,especially K₄-ψ and K₅-ψ. Yet further examples include k₁₋₁₄-ψ, k₁₋₉-ψ,and k₁₋₆-ψ, e.g. k₂-ψ, k₃-ψ, k₁₋₅-ψ and k₆-ψ especially k₄-ψ and k₅-ψ.Yet further examples include KEψ (SEQ ID NO: 15), EKEKEψ (SEQ ID NO:16), EkEkEψ (SEQ ID NO: 17), AKAAEψ (SEQ ID NO: 18), AKEKEψ (SEQ ID NO:19), and ATILEψ (SEQ ID NO: 20).

In some embodiments, U is absent.

In some embodiments, R¹ is Hy and/or R² is OH.

The peptide X* or the peptide X*—U may have the sequence:

(SEQ ID NO: 21) H[Aib]EGTFSSELATILDΨEAARDFIAWLIEHKITD; (SEQ ID NO: 22)H[Aib]EGSFTSELATILDΨEAARDFIAWLIEHKITD; (SEQ ID NO: 23)H[Aib]EGTFTSELATILDΨEAARDFIAWLIEHKITD; (SEQ ID NO: 24)H[Aib]EGTFSSELATILDΨKAARDFIAWLIEHKITD; (SEQ ID NO: 25)H[Aib]EGSFTSELATILDΨKAARDFIAWLIEHKITD; (SEQ ID NO: 26)H[Aib]EGTFTSELATILDΨKAARDFIAWLIEHKITD; (SEQ ID NO: 27)H[Aib]EGTFSSELATILDGΨAARDFIAWLIEHKITD; (SEQ ID NO: 28)H[Aib]EGSFTSELATILDGΨAARDFIAWLIEHKITD; (SEQ ID NO: 29)H[Aib]EGTFTSELATILDGΨAARDFIAWLIEHKITD; (SEQ ID NO: 30)H[Aib]EGTFSSELATILDΨLAARDFIAWLIEHKITD; (SEQ ID NO: 31)H[Aib]EGSFTSELATILDΨLAARDFIAWLIEHKITD; (SEQ ID NO: 32)H[Aib]EGTFTSELATILDΨLAARDFIAWLIEHKITD; (SEQ ID NO: 33)H[Aib]EGTFSSELATILDΨLAARDFIAWLIAHKITD; (SEQ ID NO: 34)H[Aib]EGSFTSELATILDΨLAARDFIAWLIAHKITD; (SEQ ID NO: 35)H[Aib]EGTFTSELATILDΨLAARDFIAWLIAHKITD; (SEQ ID NO: 36)H[Aib]EGTFTSELATILDΨEAARLFIAWLIEHKITD; (SEQ ID NO: 37)H[Aib]EGTFSSELATILDΨQAARDFIAWLIQHKITD; (SEQ ID NO: 38)H[Aib]EGSFTSELATILDΨQAARDFIAWLIQHKITD; (SEQ ID NO: 39)H[Aib]EGTFTSELATILDΨQAARDFIAWLIQHKITD; (SEQ ID NO: 40)H[Aib]EGTFSSELATILDΨQAARDFIAWLIEHKITD; (SEQ ID NO: 41)H[Aib]EGTFSSELATILDΨQAARDFIAWLIAHKITD; (SEQ ID NO: 42)H[Aib]EGSFTSELATILDΨQAARDFIAWLIAHKITD; (SEQ ID NO: 43)H[Aib]EGTFTSELATILDΨQAARDFIAWLIAHKITD; (SEQ ID NO: 44)H[Aib]EGSFTSELATILDΨQAARDFIAWLIEHKITD; (SEQ ID NO: 45)H[Aib]EGTFTSELATILDΨQAARDFIAWLIEHKITD; (SEQ ID NO: 46)H[Aib]EGSFTSELATILDΨQAARDFIAWLIHHKITD; (SEQ ID NO: 47)H[Aib]EGSFTSELATILDΨQAARDFIAWLIYHKITD; (SEQ ID NO: 48)H[Aib]EGSFTSELATILDΨQAARDFIAWLILHKITD; (SEQ ID NO: 49)H[Aib]EGSFTSELATILDΨQAARDFIAWLIKHKITD; (SEQ ID NO: 50)H[Aib]EGSFTSELATILDΨQAARDFIAWLIRHKITD; (SEQ ID NO: 51)H[Aib]EGSFTSELATILDΨQAARDFIAWLISHKITD; (SEQ ID NO: 52)H[Aib]EGSFTSELATILDΨQAARDFIAWLQQHKITD; (SEQ ID NO: 53)H[Aib]EGSFTSELATILDΨQAARDFIAWLYQHKITD; (SEQ ID NO: 54)H[Aib]EGSFTSELATILDΨQAARDFIAWLKQHKITD; (SEQ ID NO: 55)H[Aib]EGSFTSELATILDΨQAARDFIAWLIQQKITD; (SEQ ID NO: 56)H[Aib]EGSFTSELATILDΨQAARDFIAWLIQYKITD; (SEQ ID NO: 57)H[Aib]EGTFSSELSTILEΨQASREFIAWLIAYKITE; (SEQ ID NO: 58)H[Aib]EGTFSSELATILDEQAARDFIAWLIAHKITDkkkkkΨ; (SEQ ID NO: 59)H[Aib]EGTFTSELATILDEQAARDFIAWLIAHKITDkkkkkΨ; (SEQ ID NO: 60)H[Aib]EGSFTSELATILDEQAARDFIAWLIEHKITDkkkkkΨ; (SEQ ID NO: 61)H[Aib]EGSFTSEΨATILDEQAARDFIAWLIEHKITD; (SEQ ID NO: 62)H[Aib]EGSFTSELATILEGΨAARDFIAWLIEHKITD; (SEQ ID NO: 63)H[Aib]EGSFTSELATILDEQAAΨDFIAWLIEHKITD; (SEQ ID NO: 64)H[Aib]EGTFTSELATILDEQAAΨDFIAWLIEHKITD; (SEQ ID NO: 65)H[Aib]EGTFTSE_(Ψ)ATILDEQAARDFIAWLIEHKITD; (SEQ ID NO: 66)H[Aib]EGSFTSELATILDA_(Ψ)AARDFIAWLIEHKITD; or (SEQ ID NO: 67)H[Aib]EGSFTSELATILDAKAA_(Ψ)DFIAWLIEHKITD.

The peptide X* or the peptide X*—U may have the sequence:

(SEQ ID NO: 68) H[Aib]EGTFSSELATILD[K*]EAARDFIAWLIEHKITD;(SEQ ID NO: 69) H[Aib]EGSFTSELATILD[K*]EAARDFIAWLIEHKITD;(SEQ ID NO: 70) H[Aib]EGTFTSELATILD[K*]EAARDFIAWLIEHKITD;(SEQ ID NO: 71) H[Aib]EGTFSSELATILD[K*]KAARDFIAWLIEHKITD;(SEQ ID NO: 72) H[Aib]EGSFTSELATILD[K*]KAARDFIAWLIEHKITD;(SEQ ID NO: 73) H[Aib]EGTFTSELATILD[K*]KAARDFIAWLIEHKITD;(SEQ ID NO: 74) H[Aib]EGTFSSELATILDG[K*]AARDFIAWLIEHKITD;(SEQ ID NO: 75) H[Aib]EGSFTSELATILDG[K*]AARDFIAWLIEHKITD;(SEQ ID NO: 76) H[Aib]EGTFTSELATILDG[K*]AARDFIAWLIEHKITD;(SEQ ID NO: 77) H[Aib]EGTFSSELATILD[K*]LAARDFIAWLIEHKITD;(SEQ ID NO: 78) H[Aib]EGSFTSELATILD[K*]LAARDFIAWLIEHKITD;(SEQ ID NO: 79) H[Aib]EGTFTSELATILD[K*]LAARDFIAWLIEHKITD;(SEQ ID NO: 80) H[Aib]EGTFSSELATILD[K*]LAARDFIAWLIAHKITD;(SEQ ID NO: 81) H[Aib]EGSFTSELATILD[K*]LAARDFIAWLIAHKITD;(SEQ ID NO: 82) H[Aib]EGTFTSELATILD[K*]LAARDFIAWLIAHKITD;(SEQ ID NO: 83) H[Aib]EGTFTSELATILD[K*]EAARLFIAWLIEHKITD;(SEQ ID NO: 84) H[Aib]EGTFSSELATILD[K*]QAARDFIAWLIQHKITD;(SEQ ID NO: 85) H[Aib]EGSFTSELATILD[K*]QAARDFIAWLIQHKITD;(SEQ ID NO: 86) H[Aib]EGTFTSELATILD[K*]QAARDFIAWLIQHKITD;(SEQ ID NO: 87) H[Aib]EGTFSSELATILD[K*]QAARDFIAWLIEHKITD;(SEQ ID NO: 88) H[Aib]EGTFSSELATILD[K*]QAARDFIAWLIAHKITD;(SEQ ID NO: 89) H[Aib]EGSFTSELATILD[K*]QAARDFIAWLIAHKITD;(SEQ ID NO: 90) H[Aib]EGTFTSELATILD[K*]QAARDFIAWLIAHKITD;(SEQ ID NO: 91) H[Aib]EGSFTSELATILD[K*]QAARDFIAWLIEHKITD;(SEQ ID NO: 92) H[Aib]EGTFTSELATILD[K*]QAARDFIAWLIEHKITD;(SEQ ID NO: 93) H[Aib]EGSFTSELATILD[K*]QAARDFIAWLIHHKITD;(SEQ ID NO: 94) H[Aib]EGSFTSELATILD[K*]QAARDFIAWLIYHKITD;(SEQ ID NO: 95) H[Aib]EGSFTSELATILD[K*]QAARDFIAWLILHKITD;(SEQ ID NO: 96) H[Aib]EGSFTSELATILD[K*]QAARDFIAWLIKHKITD;(SEQ ID NO: 97) H[Aib]EGSFTSELATILD[K*]QAARDFIAWLIRHKITD;(SEQ ID NO: 98) H[Aib]EGSFTSELATILD[K*]QAARDFIAWLISHKITD;(SEQ ID NO: 99) H[Aib]EGSFTSELATILD[K*]QAARDFIAWLQQHKITD;(SEQ ID NO: 100) H[Aib]EGSFTSELATILD[K*]QAARDFIAWLYQHKITD;(SEQ ID NO: 101) H[Aib]EGSFTSELATILD[K*]QAARDFIAWLKQHKITD;(SEQ ID NO: 102) H[Aib]EGSFTSELATILD[K*]QAARDFIAWLIQQKITD;(SEQ ID NO: 103) H[Aib]EGSFTSELATILD[K*]QAARDFIAWLIQYKITD;(SEQ ID NO: 104) H[Aib]EGTFSSELSTILE[K*]QASREFIAWLIAYKITE;(SEQ ID NO: 105) H[Aib]EGTFSSELATILDEQAARDFIAWLIAHKITDkkkkk[K*];(SEQ ID NO: 106) H[Aib]EGTFTSELATILDEQAARDFIAWLIAHKITDkkkkk[K*];(SEQ ID NO: 107) H[Aib]EGSFTSELATILDEQAARDFIAWLIEHKITDkkkkk[K*];(SEQ ID NO: 108) H[Aib]EGSFTSE[K*]ATILDEQAARDFIAWLIEHKITD;(SEQ ID NO: 109) H[Aib]EGSFTSELATILEG[K*]AARDFIAWLIEHKITD;(SEQ ID NO: 110) H[Aib]EGSFTSELATILDEQAA[K*]DFIAWLIEHKITD;(SEQ ID NO: 111) H[Aib]EGTFTSELATILDEQAA[K*]DFIAWLIEHKITD;(SEQ ID NO: 112) H[Aib]EGTFTSE[K*]ATILDEQAARDFIAWLIEHKITD;(SEQ ID NO: 113) H[Aib]EGSFTSELATILDA[K*]AARDFIAWLIEHKITD; or(SEQ ID NO: 114) H[Aib]EGSFTSELATILDAKAA[K*]DFIAWLIEHKITD;

wherein K* or k* indicates an L or D lysine residue respectively inwhich the side chain is conjugated to the substituent Z¹— or Z¹Z²—.

For example, the peptide X* or the peptide X*—U may have the sequence:

(SEQ ID NO: 115)H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]EAARDFIAWLIEHKITD;(SEQ ID NO: 116)H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]EAARDFIAWLIEHKITD;(SEQ ID NO: 117)H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]EAARDFIAWLIEHKITD;(SEQ ID NO: 118)H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]KAARDFIAWLIEHKITD;(SEQ ID NO: 119)H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]KAARDFIAWLIEHKITD;(SEQ ID NO: 120)H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]KAARDFIAWLIEHKITD;(SEQ ID NO: 121)H[Aib]EGTFSSELATILDG[K([17-carboxy-heptadecanoyl]-isoGlu)]AARDFIAWLIEHKITD;(SEQ ID NO: 122)H[Aib]EGSFTSELATILDG[K([17-carboxy-heptadecanoyl]-isoGlu)]AARDFIAWLIEHKITD;(SEQ ID NO: 123)H[Aib]EGTFTSELATILDG[K([17-carboxy-heptadecanoyl]-isoGlu)]AARDFIAWLIEHKITD;(SEQ ID NO: 124)H+Aib+EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIEHKITD;(SEQ ID NO: 125)H+Aib+EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIEHKITD;(SEQ ID NO: 126)H+Aib+EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIEHKITD;(SEQ ID NO: 127)H+Aib+EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIAHKITD;(SEQ ID NO: 128)H+Aib+EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIAHKITD;(SEQ ID NO: 129)H+Aib+EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIAHKITD;(SEQ ID NO: 130)H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]EAARLFIAWLIEHKITD;(SEQ ID NO: 131)H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIQHKITD;(SEQ ID NO: 132)H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIQHKITD;(SEQ ID NO: 133)H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIQHKITD;(SEQ ID NO: 134)H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIEHKITD;(SEQ ID NO: 135)H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIAHKITD;(SEQ ID NO: 136)H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIAHKITD;(SEQ ID NO: 137)H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIAHKITD;(SEQ ID NO: 138)H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIEHKITD;(SEQ ID NO: 139)H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIEHKITD;(SEQ ID NO: 140)H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIHHKITD;(SEQ ID NO: 141)H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIYHKITD;(SEQ ID NO: 142)H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLILHKITD;(SEQ ID NO: 143)H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIKHKITD;(SEQ ID NO: 144)H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIRHKITD;(SEQ ID NO: 145)H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLISHKITD;(SEQ ID NO: 146)H[Aib]EGSFTSELATILD[K([Hexadecanoyl]-βAla)]QAARDFIAWLQQHKITD;(SEQ ID NO: 147)H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]iso-Glu-Peg3)]QAARDFIAWLYQHKITD;(SEQ ID NO: 148)H[Aib]EGSFTSELATILD[K([19-carboxy-nonadecanoyl]iso-Glu-Peg3-Peg3)]QAARDFIAWLKQHKITD; (SEQ ID NO: 149)H[Aib]EGSFTSELATILD[K([19-carboxy-nonadecanoyl]iso-Lys-Peg3-Peg3-Peg3)]QAARDFIAWLIQQKITD; (SEQ ID NO: 150)H[Aib]EGSFTSELATILD[K(Octadecanoyl)]QAARDFIAWLIQYKITD; (SEQ ID NO: 151)H[Aib]EGTFSSELSTILE[K(Hexadecanoyl-isoGlu)]QASREFIAWLIAYKITE;(SEQ ID NO: 152)H[Aib]EGTFSSELATILDEQAARDFIAWLIAHKITDkkkkkk([17-carboxy-Heptadecanoyl]-isoGlu)];(SEQ ID NO: 153)H[Aib]EGTFTSELATILDEQAARDFIAWLIAHKITDkkkkkk([17-carboxy-Heptadecanoyl]-isoGlu)];(SEQ ID NO: 154)H[Aib]EGSFTSELATILDEQAARDFIAWLIEHKITDkkkkkk([17-carboxy-Heptadecanoyl]-isoGlu)];(SEQ ID NO: 155)H[Aib]EGTFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu)]QAARDFIAWLIQHKITD;(SEQ ID NO: 156) H[Aib]EGSFTSE[K([19-carboxy-nonadecanoyl]iso-Glu-Peg3-Peg3)]ATILDEQAARDFIAWLIEHKITD; (SEQ ID NO: 157)H[Aib]EGSFTSELATILD[K([19-carboxy-nonadecanoyl]iso-Glu-Peg3-Peg3)]KAARDFIAWLIEHKITD; (SEQ ID NO: 158)H[Aib]EGSFTSELATILEG[K([19-carboxy-nonadecanoyl]iso-Glu-Peg3-Peg3)]AARDFIAWLIEHKITD; (SEQ ID NO: 159)H[Aib]EGSFTSELATILDEQAA[K([19-carboxy-nonadecanoyl]iso-Glu-Peg3-Peg3)]DFIAWLIEHKITD; (SEQ ID NO: 160)H[Aib]EGTFTSELATILDEQAA[K([19-carboxy-nonadecanoyl]iso-Glu-Peg3-Peg3)]DFIAWLIEHKITD; (SEQ ID NO: 161)H[Aib]EGTFSSELATILD[K([17-Carboxy-heptadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIQHKITD; (SEQ ID NO: 162)H[Aib]EGTFSSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIQHKITD; (SEQ ID NO: 163)H[Aib]EGTFSSELATILD[K([17-Carboxy-heptadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIEHKITD; (SEQ ID NO: 164)H[Aib]EGTFSSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIEHKITD; (SEQ ID NO: 165)H[Aib]EGTFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK)]QAARDFIAWLIQHKITD;(SEQ ID NO: 166)H[Aib]EGTFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIQHKITD; (SEQ ID NO: 167)H[Aib]EGSFTSE[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]ATILDEQAARDFIAWLIEHKITD; (SEQ ID NO: 168)H[Aib]EGTFTSE[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]ATILDEQAARDFIAWLIEHKITD; (SEQ ID NO: 169)H[Aib]EGSFTSE[K([19-carboxy-nonadecanoyl]iso-Glu-KEK-Peg3-Peg3)]ATILDEQAARDFIAWLIEHKITD; (SEQ ID NO: 170)H[Aib]EGTFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIEHKITD; (SEQ ID NO: 171)H[Aib]EGSFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIEHKITD; (SEQ ID NO: 172)H[Aib]EGSFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIAHKITD; (SEQ ID NO: 173)H[Aib]EGSFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]KAARDFIAWLIEHKITD; (SEQ ID NO: 174)H[Aib]EGSFTSELATILD[K([19-carboxy-nonadecanoyl]iso-Glu-KEK-Peg3-Peg3)]QAARDFIAWLIEHKITD; (SEQ ID NO: 175)H[Aib]EGSFTSELATILEG[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]AARDFIAWLIEHKITD; (SEQ ID NO: 176)H[Aib]EGSFTSELATILDA[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]AARDFIAWLIEHKITD; (SEQ ID NO: 177)H[Aib]EGSFTSELATILDA[K([19-carboxy-nonadecanoyl]iso-Glu-KEK-Peg3-Peg3)]AARDFIAWLIEHKITD; (SEQ ID NO: 178)H[Aib]EGSFTSELATILDEQAA[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]DFIAWLIEHKITD; (SEQ ID NO: 179)H[Aib]EGTFTSELATILDEQAA[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]DFIAWLIEHKITD; (SEQ ID NO: 180)H[Aib]EGSFTSELATILDEQAA[K([19-carboxy-nonadecanoyl]iso-Glu-KEK-Peg3-Peg3)]DFIAWLIEHKITD; (SEQ ID NO: 181)H[Aib]EGTFTSELATILDEQAA[K([19-carboxy-nonadecanoyl]iso-Glu-KEK-Peg3-Peg3)]DFIAWLIEHKITD; or (SEQ ID NO: 182)H[Aib]EGSFTSELATILDAKAA[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]DFIAWLIEHKITD.

The dual agonist may be:

(Compound 1) (SEQ ID NO: 183)Hy-H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]EAARDFIAWLIEHKITD-OH; (Compound 2) (SEQ ID NO: 184)Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]EAARDFIAWLIEHKITD-OH; (Compound 3) (SEQ ID NO: 185)Hy-H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]EAARDFIAWLIEHKITD-OH; (Compound 4) (SEQ ID NO: 186)Hy-H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]KAARDFIAWLIEHKITD-OH; (Compound 5) (SEQ ID NO: 187)Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]KAARDFIAWLIEHKITD-OH; (Compound 6) (SEQ ID NO: 188)Hy-H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]KAARDFIAWLIEHKITD-OH; (Compound 7) (SEQ ID NO: 189)Hy-H[Aib]EGTFSSELATILDG[K([17-carboxy-heptadecanoyl]-isoGlu)]AARDFIAWLIEHKITD-OH; (Compound 8) (SEQ ID NO: 190)Hy-H[Aib]EGSFTSELATILDG[K([17-carboxy-heptadecanoyl]-isoGlu)]AARDFIAWLIEHKITD-OH; (Compound 9) (SEQ ID NO: 191)Hy-H[Aib]EGTFTSELATILDG[K([17-carboxy-heptadecanoyl]-isoGlu)]AARDFIAWLIEHKITD-OH; (Compound 10) (SEQ ID NO: 192)Hy-H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIEHKITD-OH; (Compound 11) (SEQ ID NO: 193)Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIEHKITD-OH; (Compound 12) (SEQ ID NO: 194)Hy-H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIEHKITD-OH; (Compound 13) (SEQ ID NO: 195)Hy-H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIAHKITD-OH; (Compound 14) (SEQ ID NO: 196)Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIAHKITD-OH; (Compound 15) (SEQ ID NO: 197)Hy-H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIAHKITD-OH; (Compound 16) (SEQ ID NO: 198)Hy-H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]EAARLFIAWLIEHKITD-OH; (Compound 17) (SEQ ID NO: 199)Hy-H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIQHKITD-OH; (Compound 18) (SEQ ID NO: 200)Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIQHKITD-OH; (Compound 19) (SEQ ID NO: 201)Hy-H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIQHKITD-OH; (Compound 20) (SEQ ID NO: 202)Hy-H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIEHKITD-OH; (Compound 21) (SEQ ID NO: 203)Hy-H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIAHKITD-OH; (Compound 22) (SEQ ID NO: 204)Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIAHKITD-OH; (Compound 23) (SEQ ID NO: 205)Hy-H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIAHKITD-OH; (Compound 24) (SEQ ID NO: 206)Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIEHKITD-OH; (Compound 25) (SEQ ID NO: 207)Hy-H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)+QAARDFIAWLIEHKITD-OH; (Compound 26) (SEQ ID NO: 208)Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIHHKITD-OH; (Compound 27) (SEQ ID NO: 209)Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIYHKITD-OH; (Compound 28) (SEQ ID NO: 210)Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLILHKITD-OH; (Compound 29) (SEQ ID NO: 211)Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIKHKITD-OH; (Compound 30) (SEQ ID NO: 212)Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIRHKITD-OH; (Compound 31) (SEQ ID NO: 213)Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLISHKITD-OH; (Compound 32) (SEQ ID NO: 214)Hy-H[Aib]EGSFTSELATILD[K([Hexadecanoyl]-βAla)]QAARDFIAWLQQHKITD-OH;(Compound 33) (SEQ ID NO: 215)Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]iso-Glu-Peg3)]QAARDFIAWLYQHKITD-OH; (Compound 34) (SEQ ID NO: 216)Hy-H[Aib]EGSFTSELATILD[K([19-carboxy-nonadecanoyl]iso-Glu-Peg3-Peg3)]QAARDFIAWLKQHKITD-OH; (Compound 35) (SEQ ID NO: 217)Hy-H[Aib]EGSFTSELATILD[K([19-carboxy-nonadecanoyl]iso-Lys-Peg3-Peg3-Peg3)]QAARDFIAWLIQQKITD-OH; (Compound 36) (SEQ ID NO: 218)Hy-H[Aib]EGSFTSELATILD[K(Octadecanoyl)]QAARDFIAWLIQYKITD-OH;(Compound 37) (SEQ ID NO: 219) Hy-H+Aib+EGTFSSELSTILE[K(Hexadecanoyl-isoGlu)]QASREFIAWLIAYKITE-OH; (Compound 38)(SEQ ID NO: 220)Hy-H[Aib]EGTFSSELATILDEQAARDFIAWLIAHKITDkkkkkk([17-carboxy-Heptadecanoyl-isoGlu)]-[NH2]; (Compound 39) (SEQ ID NO: 221)Hy-H[Aib]EGTFTSELATILDEQAARDFIAWLIAHKITDkkkkkk([17-carboxy-Heptadecanoyl-isoGlu)]-[NH2]; (Compound 40) (SEQ ID NO: 222)Hy-H[Aib]EGSFTSELATILDEQAARDFIAWLIEHKITDkkkkkk([17-carboxy-Heptadecanoyl]-isoGlu)]-[NH2]; (Compound 41) (SEQ ID NO: 223)Hy-H[Aib]EGTFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu)+QAARDFIAWLIQHKITD-OH; (Compound 42) (SEQ ID NO: 224)Hy-H[Aib]EGSFTSE[K([19-carboxy-nonadecanoyl]iso-Glu-Peg3-Peg3)]ATILDEQAARDFIAWLIEHKITD-OH; (Compound 43) (SEQ ID NO: 225)Hy-H[Aib]EGSFTSELATILD[K([19-carboxy-nonadecanoyl]iso-Glu-Peg3-Peg3)]KAARDFIAWLIEHKITD-OH; (Compound 44) (SEQ ID NO: 226)Hy-H[Aib]EGSFTSELATILEG[K([19-carboxy-nonadecanoyl]iso-Glu-Peg3-Peg3)]AARDFIAWLIEHKITD-OH; (Compound 45) (SEQ ID NO: 227)Hy-H[Aib]EGSFTSELATILDEQAA[K([19-carboxy-nonadecanoyl]iso-Glu-Peg3-Peg3)]DFIAWLIEHKITD-OH; (Compound 46) (SEQ ID NO: 228)Hy-H[Aib]EGTFTSELATILDEQAA[K([19-carboxy-nonadecanoyl]iso-Glu-Peg3-Peg3)]DFIAWLIEHKITD-OH; (Compound 47) (SEQ ID NO: 229)Hy-H[Aib]EGTFSSELATILD[K([17-Carboxy-heptadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIQHKITD-OH; (Compound 48) (SEQ ID NO: 230)Hy-H[Aib]EGTFSSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIQHKITD-OH; (Compound 49) (SEQ ID NO: 231)Hy-H[Aib]EGTFSSELATILD[K([17-Carboxy-heptadecanoyl]-isoGlu-KEK-Peg3)+QAARDFIAWLIEHKITD-OH; (Compound 50) (SEQ ID NO: 232)Hy-H[Aib]EGTFSSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIEHKITD-OH; (Compound 51) (SEQ ID NO: 233)Hy-H[Aib]EGTFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK)]QAARDFIAWLIQHKITD-OH; (Compound 52) (SEQ ID NO: 234)Hy-H[Aib]EGTFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIQHKITD-OH; (Compound 53) (SEQ ID NO: 235)Hy-H[Aib]EGSFTSE[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]ATILDEQAARDFIAWLIEHKITD-OH; (Compound 54) (SEQ ID NO: 236)Hy-H[Aib]EGTFTSE[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]ATILDEQAARDFIAWLIEHKITD-OH; (Compound 55) (SEQ ID NO: 237)Hy-H[Aib]EGSFTSE[K([19-carboxy-nonadecanoyl]iso-Glu-KEK-Peg3-Peg3)]ATILDEQAARDFIAWLIEHKITD-OH; (Compound 56) (SEQ ID NO: 238)Hy-H[Aib]EGTFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIEHKITD-OH; (Compound 57) (SEQ ID NO: 239)Hy-H[Aib]EGSFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIEHKITD-OH; (Compound 58) (SEQ ID NO: 240)Hy-H[Aib]EGSFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIAHKITD-OH; (Compound 59) (SEQ ID NO: 241)Hy-H[Aib]EGSFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]KAARDFIAWLIEHKITD-OH; (Compound 60) (SEQ ID NO: 242)Hy-H[Aib]EGSFTSELATILD[K([19-carboxy-nonadecanoyl]iso-Glu-KEK-Peg3-Peg3)]QAARDFIAWLIEHKITD-OH; (Compound 61) (SEQ ID NO: 243)Hy-H[Aib]EGSFTSELATILEG[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]AARDFIAWLIEHKITD-OH; (Compound 62) (SEQ ID NO: 244)Hy-H[Aib]EGSFTSELATILDA[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]AARDFIAWLIEHKITD-OH; (Compound 63) (SEQ ID NO: 245)Hy-H[Aib]EGSFTSELATILDA[K([19-carboxy-nonadecanoyl]iso-Glu-KEK-Peg3-Peg3)]AARDFIAWLIEHKITD-OH; (Compound 64) (SEQ ID NO: 246)Hy-H[Aib]EGSFTSELATILDEQAA[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]DFIAWLIEHKITD-OH; (Compound 65) (SEQ ID NO: 247)Hy-H[Aib]EGTFTSELATILDEQAA[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]DFIAWLIEHKITD-OH; (Compound 66) (SEQ ID NO: 248)Hy-H[Aib]EGSFTSELATILDEQAA[K([19-carboxy-nonadecanoyl]iso-Glu-KEK-Peg3-Peg3)]DFIAWLIEHKITD-OH; (Compound 67) (SEQ ID NO: 249)Hy-H[Aib]EGTFTSELATILDEQAA[K([19-carboxy-nonadecanoyl]iso-Glu-KEK-Peg3-Peg3)]DFIAWLIEHKITD-OH; or (Compound 68) (SEQ ID NO: 250)Hy-H[Aib]EGSFTSELATILDAKAA[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]DFIAWLIEHKITD-OH.

The dual agonist may be in the form of a pharmaceutically acceptablesalt or solvate, such as a pharmaceutically acceptable acid additionsalt.

The invention also provides a composition comprising a dual agonist ofthe invention, or a pharmaceutically acceptable salt or solvate thereof,together with a carrier, excipient or vehicle. The carrier may be apharmaceutically acceptable carrier.

The composition may be a pharmaceutical composition. The pharmaceuticalcomposition may be formulated as a liquid suitable for administration byinjection or infusion. It may be formulated to achieve slow release ofthe dual agonist.

The present invention further provides a dual agonist of the inventionfor use in therapy. In yet another aspect there is provided a dualagonist of the present invention for use as a medicament. Also providedis a dual agonist of the invention for use in a method of medicaltreatment.

The invention also provides a dual agonist of the invention for use in amethod of increasing intestinal mass, improving intestinal function(especially intestinal barrier function), increasing intestinal bloodflow, or repairing intestinal damage or dysfunction, e.g., damage to theintestinal epithelium.

The invention also provides a dual agonist of the invention for use in amethod of prophylaxis or treatment of malabsorption, ulcers (e.g.,peptic ulcers, Zollinger-Ellison Syndrome, drug-induced ulcers, andulcers related to infections or other pathogens), short-bowel syndrome,cul-de-sac syndrome, inflammatory bowel disease (e.g., Crohn's diseaseand ulcerative colitis), irritable bowel syndrome (IBS), pouchitis,celiac sprue (for example arising from gluten induced enteropathy orceliac disease), tropical sprue, hypogammaglobulinemic sprue, mucositisinduced by chemotherapy or radiation therapy, diarrhea induced bychemotherapy or radiation therapy, low grade inflammation, metabolicendotoxemia, necrotising enterocolitis, primary biliary cirrhosis,hepatitis, fatty liver disease (including parental nutrition associatedgut atrophy, PNALD (Parenteral Nutrition-Associated Liver Disease),NAFLD (Non-Alcoholic Fatty Liver Disease) and NASH (Non-AlcoholicSteatohepatitis)), or gastrointestinal side-effects of inflammatoryconditions such as pancreatitis or graft versus host disease (GVHD).

The invention also provides a dual agonist of the invention for use in amethod of reducing or inhibiting weight gain, reducing gastric emptyingor intestinal transit, reducing food intake, reducing appetite, orpromoting weight loss.

The invention also provides a dual agonist of the invention for use in amethod of prophylaxis or treatment of obesity, morbid obesity,obesity-linked gallbladder disease, obesity-induced sleep apnea,inadequate glucose control, glucose tolerance, dyslipidemia (e.g.,elevated LDL levels or reduced HDL/LDL ratio), diabetes (e.g., Type 2diabetes, gestational diabetes), pre-diabetes, metabolic syndrome orhypertension.

The invention also provides a method of increasing intestinal mass,improving intestinal function (especially intestinal barrier function),increasing intestinal blood flow, or repairing intestinal damage ordysfunction in a subject in need thereof, the method comprisingadministering a dual agonist of the invention to the subject.

The invention also provides a method of prophylaxis or treatment ofmalabsorption, ulcers (e.g., peptic ulcers, Zollinger-Ellison Syndrome,drug-induced ulcers, and ulcers related to infections or otherpathogens), short-bowel syndrome, cul-de-sac syndrome, inflammatorybowel disease (Crohn's disease and ulcerative colitis), irritable bowelsyndrome (IBS), pouchitis, celiac sprue (for example arising from gluteninduced enteropathy or celiac disease), tropical sprue,hypogammaglobulinemic sprue, mucositis induced by chemotherapy orradiation therapy, diarrhea induced by chemotherapy or radiationtherapy, low grade inflammation, metabolic endotoxemia, necrotisingenterocolitis, primary biliary cirrhosis, hepatitis, fatty liver disease(including parental nutrition associated gut atrophy, PNALD (ParenteralNutrition-Associated Liver Disease), NAFLD (Non-Alcoholic Fatty LiverDisease) and NASH (Non-Alcoholic Steatohepatitis)), or gastrointestinalside-effects of inflammatory conditions such as pancreatitis or graftversus host disease (GVHD) in a subject in need thereof, the methodcomprising administering a dual agonist of the invention to the subject.

The invention also provides a method of reducing or inhibiting weightgain, reducing gastric emptying or intestinal transit, reducing foodintake, reducing appetite, or promoting weight loss in a subject in needthereof, the method comprising administering a dual agonist of theinvention to the subject.

The invention also provides a method of prophylaxis or treatment ofobesity, morbid obesity, obesity-linked gallbladder disease,obesity-induced sleep apnea, inadequate glucose control, glucosetolerance, dyslipidemia (e.g., elevated LDL levels or reduced HDL/LDLratio), diabetes (e.g., Type 2 diabetes, gestational diabetes),pre-diabetes, metabolic syndrome or hypertension in a subject in needthereof, the method comprising administering a dual agonist of theinvention to the subject.

The invention also provides the use of a dual agonist of the inventionin the preparation of a medicament for increasing intestinal mass,improving intestinal function (especially intestinal barrier function),increasing intestinal blood flow, or repairing intestinal damage ordysfunction, e.g., damage to the intestinal epithelium.

The invention also provides the use of a dual agonist of the inventionin the preparation of a medicament for prophylaxis or treatment ofmalabsorption, ulcers (e.g., peptic ulcers, Zollinger-Ellison Syndrome,drug-induced ulcers, and ulcers related to infections or otherpathogens), short-bowel syndrome, cul-de-sac syndrome, inflammatorybowel disease (Crohn's disease and ulcerative colitis), irritable bowelsyndrome (IBS), pouchitis, celiac sprue (for example arising from gluteninduced enteropathy or celiac disease), tropical sprue,hypogammaglobulinemic sprue, mucositis induced by chemotherapy orradiation therapy, diarrhea induced by chemotherapy or radiationtherapy, low grade inflammation, metabolic endotoxemia, necrotisingenterocolitis, primary biliary cirrhosis, hepatitis, fatty liver disease(including parental nutrition associated gut atrophy, PNALD (ParenteralNutrition-Associated Liver Disease), NAFLD (Non-Alcoholic Fatty LiverDisease) and NASH (Non-Alcoholic Steatohepatitis)), or gastrointestinalside-effects of inflammatory conditions such as pancreatitis or graftversus host disease (GVHD).

The invention also provides the use of a dual agonist of the inventionin the preparation of a medicament for reducing or inhibiting weightgain, reducing gastric emptying or intestinal transit, reducing foodintake, reducing appetite, or promoting weight loss.

The invention also provides the use of a dual agonist of the inventionin the preparation of a medicament for prophylaxis or treatment ofobesity, morbid obesity, obesity-linked gallbladder disease,obesity-induced sleep apnea, inadequate glucose control, glucosetolerance, dyslipidemia (e.g., elevated LDL levels or reduced HDL/LDLratio), diabetes (e.g., Type 2 diabetes, gestational diabetes),pre-diabetes, metabolic syndrome or hypertension.

A further aspect provides a therapeutic kit comprising a dual agonist,or a pharmaceutically acceptable salt or solvate thereof, according tothe invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Unless otherwise defined herein, scientific and technical terms used inthis application shall have the meanings that are commonly understood bythose of ordinary skill in the art. Generally, nomenclature used inconnection with, and techniques of, chemistry, molecular biology, celland cancer biology, immunology, microbiology, pharmacology, and proteinand nucleic acid chemistry, described herein, are those well-known andcommonly used in the art.

All patents, published patent applications and non-patent publicationsreferred to in this application are specifically incorporated byreference herein. In case of conflict, the present specification,including its specific definitions, will control.

Each embodiment of the invention described herein may be taken alone orin combination with one or more other embodiments of the invention.

Definitions

Unless specified otherwise, the following definitions are provided forspecific terms which are used in the present written description.

Throughout this specification, the word “comprise,” and grammaticalvariants thereof, such as “comprises” or “comprising,” will beunderstood to imply the inclusion of a stated integer or component, orgroup of integers or components, but not the exclusion of any otherinteger or component, or group of integers or components.

The singular forms “a,” “an,” and “the” include the plurals unless thecontext clearly dictates otherwise.

The term “including” is used to mean “including but not limited to.”“Including” and “including but not limited to” may be usedinterchangeably.

The terms “patient,” “subject” and “individual” may be usedinterchangeably and refer to either a human or a non-human animal. Theseterms include mammals such as humans, primates, livestock animals (e.g.,bovines and porcines), companion animals (e.g., canines and felines) androdents (e.g., mice and rats).

The term “solvate” in the context of the present invention refers to acomplex of defined stoichiometry formed between a solute (in casu, apeptide or pharmaceutically acceptable salt thereof according to theinvention) and a solvent. The solvent in this connection may, forexample, be water, ethanol or another pharmaceutically acceptable,typically small-molecular organic species, such as, but not limited to,acetic acid or lactic acid. When the solvent in question is water, sucha solvate is normally referred to as a hydrate.

The term “agonist” as employed in the context of the invention refers toa substance (ligand) that activates the receptor type in question.

Throughout the present description and claims the conventionalthree-letter and one-letter codes for naturally occurring amino acidsare used, i.e.,

A (Ala), G (Gly), L (Leu), I (Ile), V (Val), F (Phe), W (Trp), S (Ser),T (Thr), Y (Tyr), N (Asn), Q (Gin), D (Asp), E (Glu), K (Lys), R (Arg),H (His), M (Met), C (Cys) and P (Pro);

as well as generally accepted three-letter codes for other a-aminoacids, such as sarcosine (Sar), norleucine (Nle), α-aminoisobutyric acid(Aib), 2,3-diaminopropanoic acid (Dap), 2,4-diaminobutanoic acid (Dab)and 2,5-diaminopentanoic acid (ornithine; Orn). Such other a-amino acidsmay be shown in square brackets “[ ]” (e.g. “[Aib]”) when used in ageneral formula or sequence in the present specification, especiallywhen the rest of the formula or sequence is shown using the singleletter code. Unless otherwise specified, amino acid residues in peptidesof the invention are of the L-configuration. However, D-configurationamino acids may be incorporated. In the present context, an amino acidcode written with a small letter represents the D-configuration of saidamino acid, e.g., “k” represents the D-configuration of lysine (K).

Among sequences disclosed herein are sequences incorporating a “Hy-”moiety at the amino terminus (N-terminus) of the sequence, and either an“—OH” moiety or an “—NH₂” moiety at the carboxy terminus (C-terminus) ofthe sequence. In such cases, and unless otherwise indicated, a “Hy-”moiety at the N-terminus of the sequence in question indicates ahydrogen atom [i.e., R¹=hydrogen=Hy in the general formulas;corresponding to the presence of a free primary or secondary amino groupat the N-terminus], while an “—OH” or an “—NH₂” moiety at the C-terminusof the sequence indicates a hydroxy group [e.g., R²=OH in generalformulas; corresponding to the presence of a carboxy (COON) group at theC-terminus] or an amino group [e.g., R²=[NH₂] in the general formulas;corresponding to the presence of an amido (CONH₂) group at theC-terminus], respectively. In each sequence of the invention, aC-terminal “—OH” moiety may be substituted for a C-terminal “—NH₂”moiety, and vice-versa.

“Percent (%) amino acid sequence identity” with respect to the GLP-2polypeptide sequences is defined as the percentage of amino acidresidues in a candidate sequence that are identical to the amino acidresidues in the wild-type (human) GLP-2 sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Sequence alignment canbe carried out by the skilled person using techniques well known in theart, for example using publicly available software such as BLAST, BLAST2or Align software. For examples, see Altschul et al., Methods inEnzymology 266: 460-480 (1996) or Pearson et al., Genomics 46: 24-36,1997.

The percentage sequence identities used herein in the context of thepresent invention may be determined using these programs with theirdefault settings. More generally, the skilled worker can readilydetermine appropriate parameters for determining alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared.

Dual agonist compounds

In accordance with the present invention, the dual agonist has at leastone GLP-1 and at least one GLP-2 biological activity. Exemplary GLP-1physiological activities include reducing rate of intestinal transit,reducing rate of gastric emptying, reducing appetite, food intake orbody weight, and improving glucose control and glucose tolerance.Exemplary GLP-2 physiological activities include causing an increase inintestinal mass (e.g., of small intestine or colon), intestinal repair,and improving intestinal barrier function (i.e., reducing permeabilityof the intestine). These parameters can be assessed in in vivo assays inwhich the mass and the permeability of the intestine, or a portionthereof, is determined after a test animal has been treated with a dualagonist.

The dual agonists have agonist activity at the GLP-1 and GLP-2receptors, e.g., the human GLP-1 and GLP-2 receptors. EC₅₀ values for invitro receptor agonist activity may be used as a numerical measure ofagonist potency at a given receptor. An EC₅₀ value is a measure of theconcentration (e.g., mol/L) of a compound required to achieve half ofthat compound's maximal activity in a particular assay. A compoundhaving a numerical EC₅₀ at a particular receptor which is lower than theEC₅₀ of a reference compound in the same assay may be considered to havehigher potency at that receptor than the reference compound.

GLP-1 Activity

In some embodiments, the dual agonist has an EC₅₀ at the GLP-1 receptor(e.g., the human GLP-1 receptor) which is below 2.0 nM, below 1.5 nM,below 1.0 nM, below 0.9 nM, below 0.8 nM, below 0.7 nM, below 0.6 nM,below 0.5 nM, below 0.4 nM, below 0.3 nM, below 0.2 nM, below 0.1 nM,below 0.09 nM, below 0.08 nM, below 0.07 nM, below 0.06 nM, below 0.05nM, below 0.04 nM, e.g., when assessed using the GLP-1 receptor potencyassay described in the Examples below.

In some embodiments, the dual agonist has an EC₅₀ at the GLP-1 receptorwhich is between 0.005 and 2.5 nM, between 0.01 nM and 2.5 nM, between0.025 and 2.5 nM, between 0.005 and 2.0 nM, between 0.01 nM and 2.0 nM,between 0.025 and 2.0 nM, between 0.005 and 1.5 nM, between 0.01 nM and1.5 nM, between 0.025 and 1.5 nM, between 0.005 and 1.0 nM, between 0.01nM and 1.0 nM, between 0.025 and 1.0 nM, between 0.005 and 0.5 nM,between 0.01 nM and 0.5 nM, between 0.025 and 0.5 nM, between 0.005 and0.25 nM, between 0.01 nM and 0.25 nM, between 0.025 and 0.25 nM, e.g.,when assessed using the GLP-1 receptor potency assay described in theExamples below.

An alternative measure of GLP-1 agonist activity may be derived bycomparing the potency of a dual agonist with the potency of a known (orreference) GLP-1 agonist when both are measured in the same assay. Thus,the relative potency at the GLP-1 receptor may be defined as:

[EC₅₀(reference agonist)]/[EC₅₀(dual agonist)].

Thus, a value of 1 indicates that the dual agonist and reference agonisthave equal potency, a value of >1 indicates that the dual agonist hashigher potency (i.e., lower EC₅₀) than the reference agonist, and avalue of <1 indicates that the dual agonist has lower potency (i.e.,higher EC₅₀) than the reference agonist.

The reference GLP-1 agonist may, for example, be human GLP-1(7-37),liraglutide (NN2211; VICTOZA®), or Exendin-4, but is preferablyliraglutide.

Typically, the relative potency will be between 0.001 and 100, e.g.,

between 0.001 and 10, between 0.001 and 5, between 0.001 and 1, between0.001 and 0.5, between 0.001 and 0.1, between 0.001 and 0.05, or between0.001 and 0.01;

between 0.01 and 10, between 0.01 and 5, between 0.01 and 1, between0.01 and 0.5, between 0.01 and 0.1, or between 0.01 and 0.05;

between 0.05 and 10, between 0.05 and 5, between 0.05 and 1, between0.05 and 0.5, or between 0.05 and 0.1;

between 0.1 and 10, between 0.1 and 5, between 0.1 and 1, or between 0.1and 0.5;

between 0.5 and 10, between 0.5 and 5, or between 0.5 and 1;

between 1 and 10, or between 1 and 5;

or between 5 and 10.

The dual agonists described in the examples below have slightly lowerGLP-1 potency than liraglutide and so may, for example, have a relativepotency between 0.01 and 1, between 0.01 and 0.5 or between 0.01 and0.1.

By contrast, the dual agonists of the invention have higher potency atthe GLP-1 receptor (e.g., the human GLP-1 receptor) than wild type humanGLP-2 (hGLP-2 (1-33)) or [Gly2]-hGLP-2 (1-33) (i.e., human GLP-2 havingglycine at position 2, also known as teduglutide). Thus, the relativepotency of the dual agonists at the GLP-1 receptor compared to hGLP-2(1-33) or teduglutide is greater than 1, typically greater than 5 orgreater than 10, and may be up to 100, up to 500, or even higher.

GLP-2 Activity

In some embodiments, the dual agonist has an EC₅₀ at the GLP-2 receptor(e.g., the human GLP-2 receptor) which is below 2.0 nM, below 1.5 nM,below1.0 nM, below 0.9 nM, below 0.8 nM, below 0.7 nM, below 0.6 nM,below 0.5 nM, below 0.4 nM, below 0.3 nM, below 0.2 nM, below 0.1 nM,below 0.09 nM, below 0.08 nM, below 0.07 nM, below 0.06 nM, below 0.05nM, below 0.04 nM, below 0.03 nM, below 0.02 nM, or below 0.01 nM, e.g.,when assessed using the GLP-1 receptor potency assay described in theExamples below.

In some embodiments, the dual agonist has an EC₅₀ at the GLP-2 receptorwhich is between 0.005 and 2.0 nM, between 0.01 nM and 2.0 nM, between0.025 and 2.0 nM, between 0.005 and 1.5 nM, between 0.01 nM and 1.5 nM,between 0.025 and 1.5 nM, between 0.005 and 1.0 nM, between 0.01 nM and1.0 nM, between 0.025 and 1.0 nM, between 0.005 and 0.5 nM, between 0.01nM and 0.5 nM, between 0.025 and 0.5 nM, between 0.005 and 0.25 nM,between 0.01 nM and 0.25 nM, between 0.025 and 0.25 nM, e.g., whenassessed using the GLP-2 receptor potency assay described in theExamples below.

An alternative measure of GLP-2 agonist activity may be derived bycomparing the potency of a dual agonist with the potency of a known (orreference) GLP-2 agonist when both are measured in the same assay. Thus,the relative potency at the GLP-2 receptor may be defined as:

[EC₅₀(reference agonist)]/[EC₅₀(dual agonist)].

Thus, a value of 1 indicates that the dual agonist and reference agonisthave equal potency, a value of >1 indicates that the dual agonist hashigher potency (i.e., lower EC₅₀) than the reference agonist, and avalue of <1 indicates that the dual agonist has lower potency (i.e.,higher EC₅₀) than the reference agonist.

The reference GLP-2 agonist may, for example, be human GLP-2(1-33) orteduglutide ([Gly2]-hGLP-2 (1-33)), but is preferably teduglutide.Typically, the relative potency will be between 0.001 and 100, e.g.,

between 0.001 and 10, between 0.001 and 5, between 0.001 and 1, between0.001 and 0.5, between 0.001 and 0.1, between 0.001 and 0.05, or between0.001 and 0.01;

between 0.01 and 10, between 0.01 and 5, between 0.01 and 1, between0.01 and 0.5, between 0.01 and 0.1, or between 0.01 and 0.05;

between 0.05 and 10, between 0.05 and 5, between 0.05 and 1, between0.05 and 0.5, or between 0.05 and 0.1;

between 0.1 and 10, between 0.1 and 5, between 0.1 and 1, or between 0.1and 0.5; between 0.5 and 10, between 0.5 and 5, or between 0.5 and 1;

between 1 and 10, or between 1 and 5;

or between 5 and 10.

The dual agonists described in the examples below have slightly lowerGLP-2 potency than teduglutide and so may, for example, have a relativepotency between 0.01 and 1, between 0.01 and 0.5, or between 0.01 and0.1.

By contrast, the dual agonists of the invention have higher potency atthe GLP-2 receptor (e.g., the human GLP-2 receptor) than humanGLP-1(7-37), liraglutide (NN2211; VICTOZA®), or Exendin-4. Thus, therelative potency of the dual agonists at the GLP-2 receptor compared tohuman GLP-1(7-37), liraglutide (NN2211; VICTOZA®), or Exendin-4 isgreater than 1, typically greater than 5 or greater than 10, and may beup to 100, up to 500, or even higher (if the reference GLP-1 agonisteven exerts detectable activity at the GLP-2 receptor).

It will be understood that the absolute potencies of the dual agonistsat each receptor are much less important than the balance between theGLP-1 and GLP-2 agonist activities. Thus, it is perfectly acceptable forthe absolute GLP-1 or GLP-2 potency to be lower than that of knownagonists at those receptors, as long as the dual agonist compound exertsacceptable relative levels of potency at both receptors. Any apparentdeficiency in absolute potency can be compensated by an increased doseif required.

Substituents

The dual agonist of the present invention contains a residue LP whichcomprises a residue of Lys, Arg, Orn, Dap or Dab in which the side chainis conjugated to a substituent Z¹— or Z¹—Z²— wherein Z¹ represents amoiety CH₃—(CH₂)₁₀₋₂₂—(CO)— or HOOC—(CH₂)₁₀₋₂₂—(CO)— and Z² when presentrepresents a spacer.

The spacer Z² is selected from —Z^(S1)—, —Z^(S1)—Z^(S2)—,—Z^(S2)—Z^(S1)—, —Z^(S2)—, —Z^(S3)—, —Z^(S1)Z^(S3)—, —Z^(S2)Z^(S3)—,—Z^(S3)Z^(S1)—, —Z^(S3)Z^(S2)—, —Z^(S1)Z^(S2)—, —Z^(S1)Z^(S3)Z^(S2)—,—Z^(S2)Z^(S1)Z^(S3)—, —Z^(S2)Z^(S3)Z^(S1)—, —Z^(S3)Z^(S1)Z^(S2)—,—Z^(S3)Z^(S2)Z^(S1), Z^(S2)Z^(S3)Z^(S2)— wherein:

Z^(S1) is isoGlu, β-Ala, isoLys, or 4-aminobutanoyl;

Z^(S2) is -(Peg3)_(m)- where m is 1, 2, or 3; and

Z^(S3)— is a peptide sequence of 1-6 amino acid units selected from thegroup consisting of A, L, S, T, Y, Q, D, E, K, k, R, H, F and G.

In some embodiments, Z² is a spacer of the formula —Z^(S1)—,—Z^(S1)—Z^(S2)—, —Z^(S2)—Z^(S1), or Z^(S2), where —Z^(S1)— is isoGlu,β-Ala, isoLys, or 4-aminobutanoyl; and —Z^(S2)— is -(Peg3)_(m)- where mis 1, 2, or 3.

Without wishing to be bound by theory, it is believed that thehydrocarbon chain of Z¹ binds albumin in the blood stream, thusshielding the dual agonists of the present invention from enzymaticdegradation, which can enhance the half-life of the dual agonists.

The substituent may also modulate the potency of the dual agonists, withrespect to the GLP-2 receptor and/or the GLP-1 receptor.

The substituent Z¹— or Z¹—Z²— is conjugated to the functional group atthe distal end of the side-chain from the alpha-carbon of the relevantamino acid residue. The normal ability of the amino acid (Lys, Arg, Orn,Dab, Dap) side-chain in question to participate in interactions mediatedby that functional group (e.g. intra- and inter-molecular interactions)may therefore be reduced or completely eliminated by the presence of thesubstituent. Thus, the overall properties of the dual agonist may berelatively insensitive to changes in the actual amino acid conjugated tothe substituent. Consequently, it is believed that any of the residuesLys, Arg, Orn, Dab, or Dap may be present at any position where ψ ispermitted. However, in certain embodiments, it may be advantageous thatthe amino acid to which the substituent is conjugated is Lys or Orn.

The moiety Z¹ may be covalently bonded to the functional group in theamino acid side-chain, or alternatively may be conjugated to the aminoacid side-chain functional group via a spacer Z².

The term “conjugated” is used here to describe the covalent attachmentof one identifiable chemical moiety to another, and the structuralrelationship between such moieties. It should not be taken to imply anyparticular method of synthesis.

The bonds between Z¹,Z^(S1), Z^(S2), Z^(S3) and the amino acid sidechain to which the substituent is bound (collectively referred to hereinas ψ) are peptidic. In other words, the units may be joined by amidecondensation reactions.

Z¹ comprises a hydrocarbon chain having from 10 to 24 carbon (C) atoms,such as from 10 to 22 C atoms, e.g., from 10 to 20 C atoms. Preferably,it has at least 10 or at least 11 C atoms, and preferably it has 20 Catoms or fewer, e.g., 18 C atoms or fewer. For example, the hydrocarbonchain may contain 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Forexample, it may contain 18 or 20 carbon atoms.

In some embodiments, Z¹ is a group selected from dodecanoyl,tetradecanoyl, hexadecanoyl, octadecanoyl and eicosanoyl, preferablyhexadecanoyl, octadecanoyl or eicosanoyl, more preferably octadecanoylor eicosanoyl.

Alternative Z¹ groups are derived from long-chain saturatedα,ω-dicarboxylic acids of formula HOOC—(CH₂)₁₂₋₂₂—COOH, preferably fromlong-chain saturated α,ω-dicarboxylic acids having an even number ofcarbon atoms in the aliphatic chain. For example, Z¹ may be:13-carboxytridecanoyl, i.e. HOOC—(CH₂)₁₂—(CO)—;

15-carboxypentadecanoyl, i.e. HOOC—(CH₂)₁₄—(CO)—;

17-carboxyheptadecanoyl, i.e. HOOC—(CH₂)₁₆—(CO)—;

19-carboxynonadecanoyl, i.e. HOOC—(CH₂)₁₈—(CO)—; or

21-carboxyheneicosanoyl, i.e. HOOC—(CH₂)₂₀—(CO)—.

As mentioned above, Z¹ may be conjugated to the amino acid side-chain bya spacer Z². When present, the spacer is attached to Z¹ and to the aminoacid side-chain.

The spacer Z² has the —Z^(S1)—, —Z^(S1)—Z^(S2)—, —Z^(S2)—Z^(S1)—,—Z^(S2)—, —Z^(S3)—, —Z^(S1)Z^(S3)—, —Z^(S2)Z^(S3)—, —Z^(S3)Z^(S1)—,—Z³Z^(S2)—, —Z^(S1)Z^(S2)Z^(S3)—, —Z^(S1)Z^(S3)Z^(S2)—,—Z^(S2)Z^(S1)Z^(S3)—, —S^(S2)Z^(S3)Z^(S1)—, —Z^(S3)Z^(S1)Z^(S2)—,—Z^(S3)Z^(S2)Z^(S1)—, Z^(S2)Z^(S3)Z^(2—; where)

—Z^(S1)— is isoGlu, β-Ala, isoLys, or 4-aminobutanoyl;

—Z^(S2)— is -(Peg3)_(m)- where m is 1, 2, or 3; and

—Z^(S3)— is a peptide sequence of 1-6 amino acid units independentlyselected from the group consisting of A (Ala), L (Leu), S (Ser), T(Thr), Y (Tyr), Q (Gin), D (Asp), E (Glu), K (L-Lys), k (D-Lys), R(Arg), H (His), F (Phe) and G (Gly).

The terms “isoGlu” and “isoLys” indicate residues of amino acids whichparticipate in bonds via their side chain carboxyl or amine functionalgroups. Thus, isoGlu participates in bonds via its alpha amino and sidechain carboxyl group, while isoLys participates via its carboxyl andside chain amino groups. In the context of the present specification,the terms “γ-Glu” and “isoGlu” are used interchangeably.

The term Peg3 is used to refer to an 8-amino-3,6-dioxaoctanoyl group.

Z^(S3) may, for example, be 3 to 6 amino acids in length, i.e., 3, 4, 5or 6 amino acids in length.

In some embodiments, the amino acids of Z^(S3) are independentlyselected from K, k, E, A, T, I and L, e.g., from K, k, E and A, e.g.,from K, k and E.

Typically, Z^(S3) includes at least one charged amino acid (K, k, R orE, e.g. K, k or E) and preferably two or more charged amino acids. Insome embodiments it includes at least 2 positively charged amino acids(K, k or R, especially K or k), or at least 1 positively charged aminoacid (K, k or R, especially K or k) and at least one negatively chargedamino acid (E). In some embodiments, all amino acid residues of Z^(S3)are charged. For example, Z^(S3) may be a chain of alternatelypositively and negatively charged amino acids.

Examples of Z^(S3) moieties include KEK (SEQ ID NO: 8), EKEKEK (SEQ IDNO: 9), kkkkkk (SEQ ID NO: 10), EkEkEk (SEQ ID NO: 11), AKAAEK (SEQ IDNO: 12), AKEKEK (SEQ ID NO: 13) and ATILEK (SEQ ID NO: 14).

Without being bound by theory, it is believed that the incorporation ofZ^(S3) into the linker between the fatty acid chain and the peptidebackbone may increase the half-life of the dual agonist by enhancing itsaffinity for serum albumin.

In some embodiments, —Z²— is —Z^(S1)— or —Z^(S1)—Z^(S2)—; in otherwords, —Z²— is selected from:

isoGlu(Peg3)₀₋₃;

β-Ala(Peg3)₀₋₃;

isoLys(Peg3)₀₋₃; and

4-aminobutanoyl(Peg3)₀₋₃.

Thus, certain examples of substituents Z¹— include

[Dodecanoyl], [Tetradecanoyl], [Hexadecanoyl], [Octadecanoyl],[Eicosanoyl],

[13-Carboxy-tridecanoyl], [15-Carboxy-pentadecanoyl],[17-Carboxy-heptadecanoyl], [19-Carboxy-nonadecanoyl],[21-carboxy-heneicosanoyl].

More broadly, —Z_(S2)— may be —Z^(S1)—, —Z^(S1)—Z^(S2)—,—Z^(S3)—Z^(S1)—, —Z^(S1)—Z^(S3)—, —Z^(S1)—Z^(S3)—Z^(S2),—Z^(S3)—Z^(S2)—Z^(S1)—. Thus, —Z²— may be selected from the groupconsisting of:

isoGlu(Peg3)₀₋₃;

β-Ala(Peg3)₀₋₃;

isoLys(Peg3)₀₋₃;

4-aminobutanoyl(Peg3)₀₋₃;

isoGlu(KEK)(Peg3)₀₋₃;

β-Ala(KEK)(Peg3)₀₋₃;

isoLys(KEK)(Peg3)₀₋₃;

4-aminobutanoyl(KEK)(Peg3)₀₋₃;

KEK(isoGlu);

KEK(β-Ala);

KEK(isoLys);

KEK(4-aminobutanoyl);

isoGlu(KEK);

β-Ala(KEK);

isoLys(KEK);

4-aminobutanoyl(KEK);

KEK(isoGlu)(Peg3)₀₋₃;

KEK(β-Ala)(Prg3)₀₋₃;

KEK(isoLys)(Peg3)₀₋₃; and

KEK(4-aminobutanoyl)(Peg3)₀₋₃;

Certain examples of substituents Z¹—Z²— include:

[Dodecanoyl]-isoGlu, [Tetradecanoyl]-isoGlu, [Hexadecanoyl]-isoGlu,[Octadecanoyl]-isoGlu, [Eicosanoyl]-isoGlu,

[Hexadecanoyl]-βAla, [Octadecanoyl]-βAla, [Eicosanoyl]-βAla,[Tetradecanoyl]-βAla, [Dodecanoyl]-βAla,

[Dodecanoyl]isoGlu-Peg3, [Tetradecanoyl]-isoGlu-Peg3,[Hexadecanoyl]isoGlu-Peg3, [Octadecanoyl]-isoGlu-Peg3,[Eicosanoyl]-isoGlu-Peg3,

[Dodecanoyl]-βAla-Peg3, [Tetradecanoyl]-βAla-Peg3,[Hexadecanoyl]-βAla-Peg3, [Octadecanoyl]-βAla-Peg3,[Eicosanoyl]-βAla-Peg3,

[Dodecanoyl]-isoGlu-Peg3-Peg3, [Tetradecanoyl]-isoGlu-Peg3-Peg3,[Hexadecanoyl]isoGlu-Peg3-Peg3, [Octadecanoyl]-isoGlu-Peg3-Peg3,[Eicosanoyl]isoGlu-Peg3-Peg3,

[Dodecanoyl]-βAla-Peg3-Peg3, [Tetradecanoyl]-βAla-Peg3-Peg3,[Hexadecanoyl]-βAla-Peg3-Peg3, [Octadecanoyl]-βAla-Peg3-Peg3,[Eicosanoyl]-βAla-Peg3-Peg3,

[Dodecanoyl]-isoGlu-Peg3-Peg3-Peg3,[Tetradecanoyl]-isoGlu-Peg3-Peg3-Peg3,[Hexadecanoyl]-isoGlu-Peg3-Peg3-Peg3,[Octadecanoyl]isoGlu-Peg3-Peg3-Peg3, [Eicosanoyl]isoGlu-Peg3-Peg3-Peg3,

[Dodecanoyl]-βAla-Peg3-Peg3-Peg3, [Tetradecanoyl]-βAla-Peg3-Peg3-Peg3,[Hexadecanoyl]-βAla-Peg3-Peg3-Peg3, [Octadecanoyl]-βAla-Peg3-Peg3-Peg3,[Eicosanoyl]-βAla-Peg3-Peg3-Peg3,

[Dodecanoyl]-isoLys, [Tetradecanoyl]-isoLys, [Hexadecanoyl]-isoLys,[Octadecanoyl]-isoLys, [Eicosanoyl]-isoLys,

[Hexadecanoyl]-[4-aminobutanoyl], [Octadecanoyl]-[4-aminobutanoyl],[Eicosanoyl]-[4-aminobutanoyl], [Tetradecanoyl]-[4-aminobutanoyl],[Dodecanoyl]-[4-aminobutanoyl],

[Dodecanoyl]isoLys-Peg3, [Tetradecanoyl]-isoLys-Peg3,[Hexadecanoyl]-isoLys-Peg3, [Octadecanoyl]-isoLys-Peg3,[Eicosanoyl]-isoLys-Peg3,

[Dodecanoyl]-[4-aminobutanoyl]-Peg3,[Tetradecanoyl]-[4-aminobutanoyl]-Peg3,[Hexadecanoyl]-[4-aminobutanoyl]-Peg3,

[Octadecanoyl]-[4-aminobutanoyl]-Peg3,[Eicosanoyl]-[4-aminobutanoyl]-Peg3,

[Dodecanoyl]-isoLys-Peg3-Peg3, [Tetradecanoyl]-isoLys-Peg3-Peg3,[Hexadecanoyl]-isoLys-Peg3-Peg3, [Octadecanoyl]-isoLys-Peg3-Peg3,[Eicosanoyl]-isoLys-Peg3-Peg3,

[Dodecanoyl]-[4-aminobutanoyl]-Peg3-Peg3, [Tetradecanoyl]-[4-aminobutanoyl]-Peg3-Peg3, [Hexadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3,[Octadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3,[Eicosanoyl]-[4-aminobutanoyl]-Peg3-Peg3,

[Dodecanoyl]-isoLys-Peg3-Peg3-Peg3,[Tetradecanoyl]-isoLys-Peg3-Peg3-Peg3,[Hexadecanoyl]-isoLys-Peg3-Peg3-Peg3,[Octadecanoyl]-isoLys-Peg3-Peg3-Peg3,[Eicosanoyl]-isoLys-Peg3-Peg3-Peg3,

[Dodecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,[Tetradecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,[Hexadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,[Octadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,[Eicosanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,

[13-carboxy-tridecanoyl]-isoGlu, [15-carboxy-Pentadecanoyl]-isoGlu,[17-carboxy-Heptadecanoyl]-isoGlu, [19-carboxy-Nonadecanoyl]-isoGlu,[21-carboxy-heneicosanoyl]-isoGlu,

[17-carboxy-Heptadecanoyl]-βAla, [19-carboxy-Nonadecanoyl]-βAla,[21-carboxy-heneicosanoyl]-βAla, [15-carboxy-Pentadecanoyl]-βAla,[13-carboxy-tridecanoyl]-βAla,

[13-carboxy-tridecanoyl]-isoGlu-Peg3,[15-carboxy-Pentadecanoyl]-isoGlu-Peg3,[17-carboxy-Heptadecanoyl]-isoGlu-Peg3,[19-carboxy-Nonadecanoyl]-isoGlu-Peg3,[21-carboxy-heneicosanoyl]-isoGlu-Peg3,

[13-carboxy-tridecanoyl]-βAla-Peg3,[15-carboxy-Pentadecanoyl]-βAla-Peg3,[17-carboxy-Heptadecanoyl]-βAla-Peg3,[19-carboxy-Nonadecanoyl]-βAla-Peg3,[21-carboxy-heneicosanoyl]-βAla-Peg3,

[13-carboxy-tridecanoyl]-isoGlu-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-isoGlu-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-isoGlu-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-isoGlu-Peg3-Peg3,[21-carboxy-heneicosanoyl]-isoGlu-Peg3-Peg3,

[13-carboxy-tridecanoyl]-βAla-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-βAla-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-βAla-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-βAla-Peg3-Peg3,[21-carboxy-heneicosanoyl]-βAla-Peg3-Peg3,

[13-carboxy-tridecanoyl]-isoGlu-Peg3-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-isoGlu-Peg3-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-isoGlu-Peg3-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-isoGlu-Peg3-Peg3-Peg3,[21-carboxy-heneicosanoyl]-isoGlu-Peg3-Peg3-Peg3,

[13-carboxy-tridecanoyl]-βAla-Peg3-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-βAla-Peg3-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-βAla-Peg3-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-βAla-Peg3-Peg3-Peg3,[21-carboxy-heneicosanoyl]-βAla-Peg3-Peg3-Peg3,

[13-carboxy-tridecanoyl]-isoLys, [15-carboxy-Pentadecanoyl]-isoLys,[17-carboxy-Heptadecanoyl]-isoLys, [19-carboxy-Nonadecanoyl]-isoLys,[21-carboxy-heneicosanoyl]-isoLys,

[17-carboxy-Heptadecanoyl]-[4-aminobutanoyl],[19-carboxy-Nonadecanoyl]-[4-aminobutanoyl],[21-carboxy-heneicosanoyl]-[4-aminobutanoyl],[15-carboxy-Pentadecanoyl]-[4-aminobutanoyl],[13-carboxy-tridecanoyl]-[4-aminobutanoyl],

[13-carboxy-tridecanoyl]-isoLys-Peg3,[15-carboxy-Pentadecanoyl]-isoLys-Peg3,[17-carboxy-Heptadecanoyl]-isoLys-Peg3,[19-carboxy-Nonadecanoyl]-isoLys-Peg3,[21-carboxy-heneicosanoyl]-isoLys-Peg3,

[13-carboxy-tridecanoyl]-[4-aminobutanoyl]-Peg3,[15-carboxy-Pentadecanoyl]-[4-aminobutanoyl]-Peg3,[17-carboxy-Heptadecanoyl]-[4-aminobutanoyl]-Peg3,[19-carboxy-Nonadecanoyl]-βAla-Peg3,[21-carboxy-heneicosanoyl]-βAla-Peg3,

[13-carboxy-tridecanoyl]-isoLys-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-isoLys-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-isoLys-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-isoLys-Peg3-Peg3,[21-carboxy-heneicosanoyl]-isoLys-Peg3-Peg3,

[13-carboxy-tridecanoyl]-[4-aminobutanoyl]-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3,[17-carboxy-Heptadecanoy]-[4-aminobutanoyl]-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3,[21-carboxy-heneicosanoyl]-[4-aminobutanoyl]-Peg3-Peg3,

[13-carboxy-tridecanoyl]-isoLys-Peg3-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-isoLys-Peg3-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-isoLys-Peg3-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-isoLys-Peg3-Peg3-Peg3,[21-carboxy-heneicosanoyl]-isoLys-Peg3-Peg3-Peg3,

[13-carboxy-tridecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3 and[21-carboxy-heneicosanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3.

Further examples of substituents Z¹—Z²— include:

[Dodecanoyl]-isoLys, [Tetradecanoyl]-isoLys, [Hexadecanoyl]-isoLys,[Octadecanoyl]-isoLys, [Eicosanoyl]-isoLys,

[Hexadecanoyl]-[4-aminobutanoyl], [Octadecanoyl]-[4-aminobutanoyl],[Eicosanoyl]-[4-aminobutanoyl], [Tetradecanoyl]-[4-aminobutanoyl],[Dodecanoyl]-[4-aminobutanoyl],

[Hexadecanoyl]-KEK (SEQ ID NO: 251), [Octadecanoyl]-KEK (SEQ ID NO:252), [Eicosanoyl]-KEK (SEQ ID NO: 253), [Tetradecanoyl]-KEK (SEQ ID NO:254), [Dodecanoyl]-KEK (SEQ ID NO: 255),

[Dodecanoyl]-Peg3, [Tetradecanoyl]-Peg3, [Hexadecanoyl]-Peg3,

[Octadecanoyl]-Peg3, [Eicosanoyl]-Peg3,

[Dodecanoyl]-Peg3-Peg3, [Tetradecanoyl]-Peg3-Peg3,

[Hexadecanoyl]-Peg3-Peg3, [Octadecanoyl]-Peg3-Peg3,[Eicosanoyl]-Peg3-Peg3,

[Dodecanoyl]-Peg3-Peg3-Peg3, [Tetradecanoyl]-Peg3-Peg3-Peg3,[Hexadecanoyl]-Peg3-Peg3-Peg3, [Octadecanoyl]-Peg3-Peg3-Peg3,[Eicosanoyl]-Peg3-Peg3-Peg3,

[Dodecanoyl]-isoLys-Peg3, [Tetradecanoyl]-isoLys-Peg3,[Hexadecanoyl]-isoLys-Peg3, [Octadecanoyl]-isoLys-Peg3,[Eicosanoyl]-isoLys-Peg3,

[Dodecanoyl]-[4-aminobutanoyl]-Peg3,[Tetradecanoyl]-[4-aminobutanoyl]-Peg3,[Hexadecanoyl]-[4-aminobutanoyl]-Peg3,[Octadecanoyl]-[4-aminobutanoyl]-Peg3,[Eicosanoyl]-[4-aminobutanoyl]-Peg3,

[Dodecanoyl]-KEK-Peg3 (SEQ ID NO: 256), [Tetradecanoyl]-KEK-Peg3 (SEQ IDNO: 257), [Hexadecanoyl]-KEK-Peg3 (SEQ ID NO: 258),[Octadecanoyl]-KEK-Peg3 (SEQ ID NO: 259), [Eicosanoyl]-KEK-Peg3 (SEQ IDNO: 260),

[Dodecanoyl]-isoLys-Peg3-Peg3, [Tetradecanoyl]-isoLys-Peg3-Peg3,[Hexadecanoyl]-isoLys-Peg3-Peg3, [Octadecanoyl]-isoLys-Peg3-Peg3,[Eicosanoyl]-isoLys-Peg3-Peg3,

[Dodecanoyl]-[4-aminobutanoyl]-Peg3-Peg3,[Tetradecanoyl]-[4-aminobutanoyl]-Peg3-Peg3,[Hexadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3,[Octadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3,[Eicosanoyl]-[4-aminobutanoyl]-Peg3-Peg3,

[Dodecanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 261),[Tetradecanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 262),[Hexadecanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 263), [Octadecanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 264), [Eicosanoyl]-KEK-Peg3-Peg3 (SEQ ID NO:265),

[Dodecanoyl]-isoLys-Peg3-Peg3-Peg3,[Tetradecanoyl]-isoLys-Peg3-Peg3-Peg3,[Hexadecanoyl]-isoLys-Peg3-Peg3-Peg3,[Octadecanoyl]-isoLys-Peg3-Peg3-Peg3,[Eicosanoyl]-isoLys-Peg3-Peg3-Peg3,

[Dodecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,[Tetradecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,[Hexadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,[Octadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,[Eicosanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,

[Dodecanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 266),[Tetradecanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 267),[Hexadecanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 268),[Octadecanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 269),[Eicosanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 270),

[Dodecanoyl]-isoGlu-KEK-Peg3 (SEQ ID NO: 271),[Tetradecanoyl]-isoGlu-KEK-Peg3 (SEQ ID NO: 272),[Hexadecanoyl]-isoGlu-KEK-Peg3 (SEQ ID NO: 273),[Octadecanoyl]-isoGlu-KEK-Peg3 (SEQ ID NO: 274),[Eicosanoyl]-isoGlu-KEK-Peg3 (SEQ ID NO: 275),

[Dodecanoyl]-[4-aminobutanoyl]-KEK-Peg3 (SEQ ID NO: 276),[Tetradecanoyl]-[4-aminobutanoyl]-KEK-Peg3 (SEQ ID NO: 277),[Hexadecanoyl]-[4-aminobutanoyl]-KEK-Peg3 (SEQ ID NO: 278),[Octadecanoyl]-[4-aminobutanoyl]-KEK-Peg3 (SEQ ID NO: 279),[Eicosanoyl]-[4-aminobutanoyl]-KEK-Peg3 (SEQ ID NO: 280),

[Dodecanoyl]-isoLys-KEK-Peg3 (SEQ ID NO: 281),[Tetradecanoyl]-isoLys-KEK-Peg3 (SEQ ID NO: 282),[Hexadecanoyl]-isoLys-KEK-Peg3 (SEQ ID NO: 283),[Octadecanoyl]-isoLys-KEK-Peg3 (SEQ ID NO: 284),[Eicosanoyl]-isoLys-KEK-Peg3 (SEQ ID NO: 285),

[Dodecanoyl]-Ala-KEK-Peg3 (SEQ ID NO: 286), [Tetradecanoyl]-Ala-KEK-Peg3(SEQ ID NO: 287), [Hexadecanoyl]-Ala-KEK-Peg3 (SEQ ID NO: 288),[Octadecanoyl]-Ala-KEK-Peg3 (SEQ ID NO: 289), [Eicosanoyl]-Ala-KEK-Peg3,(SEQ ID NO: 290)

[Dodecanoyl]-isoGlu-KEK-Peg3-Peg3 (SEQ ID NO: 291),[Tetradecanoyl]-isoGlu-KEK-Peg3-Peg3 (SEQ ID NO: 292),[Hexadecanoyl]-isoGlu-KEK-Peg3-Peg3 (SEQ ID NO: 293),[Octadecanoyl]-isoGlu-KEK-Peg3-Peg3 (SEQ ID NO: 294),[Eicosanoyl]-isoGlu-KEK-Peg3-Peg3 (SEQ ID NO: 295),

[Dodecanoyl]-βAla-KEK-Peg3-Peg3 (SEQ ID NO: 296),[Tetradecanoyl]-βAla-KEK-Peg3-Peg3 (SEQ ID NO: 297),[Hexadecanoyl]-βAla-KEK-Peg3-Peg3 (SEQ ID NO: 298),[Octadecanoyl]-βAla-KEK-Peg3-Peg3 (SEQ ID NO: 299),[Eicosanoyl]-βAla-KEK-Peg3-Peg3 (SEQ ID NO: 300),

[Dodecanoyl]-isoLys-KEK-Peg3-Peg3 (SEQ ID NO: 301),[Tetradecanoyl]-isoLys-KEK-Peg3-Peg3 (SEQ ID NO: 302),[Hexadecanoyl]-isoLys-KEK-Peg3-Peg3 (SEQ ID NO: 303),[Octadecanoyl]-isoLys-KEK-Peg3-Peg3 (SEQ ID NO: 304),[Eicosanoyl]-isoLys-KEK-Peg3-Peg3 (SEQ ID NO: 305),

[Dodecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 306),[Tetradecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 307),[Hexadecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 308),[Octadecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 309),[Eicosanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 310),

[Dodecanoyl]-isoGlu-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 311),[Tetradecanoyl]-isoGlu-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 312),[Hexadecanoyl]-isoGlu-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 313),

[Octadecanoyl]-isoGlu-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 314),[Eicosanoyl]-isoGlu-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 315),

[Dodecanoyl]-βAla-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 316),[Tetradecanoyl]-βAla-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 317),[Hexadecanoyl]-βAla-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 318),[Octadecanoyl]-βAla-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 319),[Eicosanoyl]-βAla-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 320),

[Dodecanoyl]-isoLys-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 321),[Tetradecanoyl]-isoLys-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 322),[Hexadecanoyl]-isoLys-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 323),

[Octadecanoyl]-isoLys-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 324),[Eicosanoyl]-isoLys-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 325),

[Dodecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 326),[Tetradecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 327),[Hexadecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 328),[Octadecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 329),[Eicosanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 330),

[Dodecanoyl]-KEK-isoGlu-Peg3 (SEQ ID NO: 331),[Tetradecanoyl]-KEK-isoGlu-Peg3 (SEQ ID NO: 332),[Hexadecanoyl]-KEK-isoGlu-Peg3 (SEQ ID NO: 333),[Octadecanoyl]-KEK-isoGlu-Peg3 (SEQ ID NO: 334),[Eicosanoyl]-KEK-isoGlu-Peg3 (SEQ ID NO: 335),

[Dodecanoyl]-KEK-Ala-βPeg3 (SEQ ID NO: 336),[Tetradecanoyl]-KEK-βAla-Peg3 (SEQ ID NO: 337),[Hexadecanoyl]-KEK-βAla-Peg3 (SEQ ID NO: 338),[Octadecanoyl]-KEK-βAla-Peg3, (SEQ ID NO: 339)[Eicosanoyl]-KEK-βAla-Peg3 (SEQ ID NO: 340),

[Dodecanoyl]-KEK-[4-aminobutanoyl]-Peg3 (SEQ ID NO: 341),[Tetradecanoyl]-KEK-[4-aminobutanoyl]-Peg3 (SEQ ID NO: 342),[Hexadecanoyl]-KEK-[4-aminobutanoyl]-Peg3 (SEQ ID NO: 343),[Octadecanoyl]-KEK-[4-aminobutanoyl]-Peg3 (SEQ ID NO: 344),[Eicosanoyl]-KEK-[4-aminobutanoyl]-Peg3 (SEQ ID NO: 345),

[Dodecanoyl]-KEK-isoLys-Peg3 (SEQ ID NO: 346),[Tetradecanoyl]-KEK-isoLys-Peg3 (SEQ ID NO: 347),[Hexadecanoyl]-KEK-isoLys-Peg3 (SEQ ID NO: 348),[Octadecanoyl]-KEK-isoLys-Peg3 (SEQ ID NO: 349),[Eicosanoyl]-KEK-isoLys-Peg3 (SEQ ID NO: 350),

[Dodecanoyl]-KEK-isoGlu-Peg3-Peg3 (SEQ ID NO: 351),[Tetradecanoyl]-KEK-isoGlu-Peg3-Peg3 (SEQ ID NO: 352),[Hexadecanoyl]-KEK-isoGlu-Peg3-Peg3 (SEQ ID NO: 353),[Octadecanoyl]-KEK-isoGlu-Peg3-Peg3 (SEQ ID NO: 354),[Eicosanoyl]-KEK-isoGlu-Peg3-Peg3 (SEQ ID NO: 355),

[Dodecanoyl]-KEK-βAla-Peg3-Peg3 (SEQ ID NO: 356),[Tetradecanoyl]-KEK-βAla-Peg3-Peg3 (SEQ ID NO: 357),[Hexadecanoyl]-KEK-βAla-Peg3-Peg3 (SEQ ID NO: 358),[Octadecanoyl]-KEK-βAla-Peg3-Peg3 (SEQ ID NO: 359),[Eicosanoyl]-βAla-KEK-Peg3-Peg3 (SEQ ID NO: 360),

[Dodecanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3 (SEQ ID NO: 361),[Tetradecanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3 (SEQ ID NO: 362),[Hexadecanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3 (SEQ ID NO: 363),[Octadecanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3 (SEQ ID NO: 364),[Eicosanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3 (SEQ ID NO: 365),

[Dodecanoyl]-KEK-isoLys-Peg3-Peg3 (SEQ ID NO: 366),[Tetradecanoyl]-KEK-isoLys-Peg3-Peg3 (SEQ ID NO: 367),[Hexadecanoyl]-KEK-isoLys-Peg3-Peg3 (SEQ ID NO: 368),[Octadecanoyl]-KEK-isoLys-Peg3-Peg3 (SEQ ID NO: 369),[Eicosanoyl]-KEK-isoLys-Peg3-Peg3 (SEQ ID NO: 370),

[Dodecanoyl]-KEK-isoGlu-Peg3-Peg3-Peg3 (SEQ ID NO: 371),[Tetradecanoyl]-KEK-isoGlu-Peg3-Peg3-Peg3 (SEQ ID NO: 372),[Hexadecanoyl]-KEK-isoGlu-Peg3-Peg3-Peg3 (SEQ ID NO: 373),

[Octadecanoyl]-KEK-isoGlu-Peg3-Peg3-Peg3 (SEQ ID NO: 374),[Eicosanoyl]-KEK-isoGlu-Peg3-Peg3-Peg3 (SEQ ID NO: 375),

[Dodecanoyl]-KEK-βAla-Peg3-Peg3-Peg3 (SEQ ID NO: 376),[Tetradecanoyl]-KEK-βAla-Peg3-Peg3-Peg3 (SEQ ID NO: 377),[Hexadecanoyl]-βAla-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 378),

[Octadecanoyl]-KEK-βAla-Peg3-Peg3-Peg3 (SEQ ID NO: 379),[Eicosanoyl]-KEK-βAla-Peg3-Peg3-Peg3 (SEQ ID NO: 380),

[Dodecanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3-Peg3 (SEQ ID NO: 381),[Tetradecanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3-Peg3 (SEQ ID NO: 382),[Hexadecanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3-Peg3 (SEQ ID NO: 383),[Octadecanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3-Peg3 (SEQ ID NO: 384),[Eicosanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3-Peg3 (SEQ ID NO: 385),

[Dodecanoyl]-KEK-isoLys-Peg3-Peg3-Peg3 (SEQ ID NO: 386),[Tetradecanoyl]-KEK-isoLys-Peg3-Peg3-Peg3 (SEQ ID NO: 387),[Hexadecanoyl]-KEK-isoLys-Peg3-Peg3-Peg3 (SEQ ID NO: 388),

[Octadecanoyl]-KEK-isoLys-Peg3-Peg3-Peg3 (SEQ ID NO: 389),[Eicosanoyl]-KEK-isoLys-Peg3-Peg3-Peg3 (SEQ ID NO: 390),

[13-carboxy-tridecanoyl]-isoGlu, [15-carboxy-Pentadecanoyl]-isoGlu,[17-carboxy-Heptadecanoyl]-isoGlu,

[19-carboxy-Nonadecanoyl]-isoGlu,[21-carboxy-hen21-carboxy-heneicosanoyl]-isoGlu,

[17-carboxy-Heptadecanoyl]-Ala, [19-carboxy-Nonadecanoyl]-Ala,[21-carboxy-heneicosanoyl]-Ala, [15-carboxy-Pentadecanoyl]-Ala,[13-carboxy-triclecanoyl]-Ala,

[13-carboxy-tridecanoyl]-isoLys, [15-carboxy-Pentadecanoyl]-isoLys,[17-carboxy-Heptadecanoyl]-isoLys,

[19-carboxy-Nonadecanoyl]-isoLys, [21-carboxy-heneicosanoyl]-isoLys,

[17-carboxy-Heptadecanoyl]-[4-aminobutanoyl],[19-carboxy-Nonadecanoyl]-[4-am inobutanoyl],[21-carboxy-heneicosanoyl]-[4-aminobutanoyl],[15-carboxy-Pentadecanoyl]-[4-aminobutanoyl],[13-carboxy-triclecanoyl]-[4-aminobutanoyl],

[17-carboxy-Heptadecanoyl]-KEK (SEQ ID NO: 391),[19-carboxy-Nonadecanoyl]-KEK (SEQ ID NO: 392),[21-carboxy-heneicosanoyl]-KEK (SEQ ID NO: 393),[15-carboxy-Pentadecanoyl]-KEK (SEQ ID NO: 394),[13-carboxy-tridecanoyl]-KEK (SEQ ID NO: 395),

[13-carboxy-tridecanoyl]-Peg3, [15-carboxy-Pentadecanoyl]-Peg3,[17-carboxy-Heptadecanoyl]-Peg3, [19-carboxy-Nonadecanoyl]-Peg3,[21-carboxy-heneicosanoyl]-Peg3,

[13-carboxy-tridecanoyl]-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-Peg3-Peg3,[21-carboxy-heneicosanoyl]-Peg3-Peg3,

[13-carboxy-tridecanoyl]-Peg3-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-Peg3-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-Peg3-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-Peg3-Peg3-Peg3,[21-carboxy-heneicosanoyl]-Peg3-Peg3-Peg3,

[13-carboxy-tridecanoyl]-isoGlu-Peg3,[15-carboxy-Pentadecanoyl]-isoGlu-Peg3,[17-carboxy-Heptadecanoyl]-isoGlu-Peg3,[19-carboxy-Nonadecanoyl]-isoGlu-Peg3,[21-carboxy-heneicosanoyl]-isoGlu-Peg3,

[13-carboxy-tridecanoyl]-βAla-Peg3,[15-carboxy-Pentadecanoyl]-βAla-Peg3,[17-carboxy-Heptadecanoyl]-βAla-Peg3,[19-carboxy-Nonadecanoyl]-βAla-Peg3,[21-carboxy-heneicosanoyl]-βAla-Peg3,

[13-carboxy-tridecanoyl]-isoLys-Peg3,[15-carboxy-Pentadecanoyl]-isoLys-Peg3,[17-carboxy-Heptadecanoyl]-isoLys-Peg3,[19-carboxy-Nonadecanoyl]-isoLys-Peg3,[21-carboxy-heneicosanoyl]-isoLys-Peg3,

[13-carboxy-tridecanoyl]-[4-aminobutanoyl]-Peg3,[15-carboxy-Pentadecanoyl]-[4-aminobutanoyl]-Peg3,[17-carboxy-Heptadecanoyl]-[4-aminobutanoyl]-Peg3,[19-carboxy-Nonadecanoyl]-[4-aminobutanoyl]-Peg3,[21-carboxy-heneicosanoyl]-[4-aminobutanoyl]-Peg3,

[13-carboxy-tridecanoyl]-KEK-Peg3 (SEQ ID NO: 396),[15-carboxy-Pentadecanoyl]-KEK-Peg3 (SEQ ID NO: 397),[17-carboxy-Heptadecanoyl]-KEK-Peg3 (SEQ ID NO: 398),[19-carboxy-Nonadecanoyl]-KEK-Peg3 (SEQ ID NO: 399),[21-carboxy-heneicosanoyl]-KEK-Peg3 (SEQ ID NO: 400),

[13-carboxy-tridecanoyl]-isoGlu-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-isoGlu-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-isoGlu-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-isoGlu-Peg3-Peg3,[21-carboxy-heneicosanoyl]-isoGlu-Peg3-Peg3,

[13-carboxy-tridecanoyl]-βAla-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-βAla-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-βAla-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-βAla-Peg3-Peg3,[21-carboxy-heneicosanoyl]-βAla-Peg3-Peg3,

[13-carboxy-tridecanoyl]-isoLys-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-isoLys-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-isoLys-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-isoLys-Peg3-Peg3,[21-carboxy-heneicosanoyl]-isoLys-Peg3-Peg3,

[13-carboxy-tridecanoyl]-[4-aminobutanoyl]-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3,[21-carboxy-heneicosanoyl]-[4-aminobutanoyl]-Peg3-Peg3,

[13-carboxy-tridecanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 401),[15-carboxy-Pentadecanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 402),[17-carboxy-Heptadecanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 403),[19-carboxy-Nonadecanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 404),[21-carboxy-heneicosanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 405),

[13-carboxy-tridecanoyl]-isoGlu-Peg3-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-isoGlu-Peg3-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-isoGlu-Peg3-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-isoGlu-Peg3-Peg3-Peg3,[21-carboxy-heneicosanoyl]-isoGlu-Peg3-Peg3-Peg3,

[13-carboxy-tridecanoyl]-βAla-Peg3-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-βAla-Peg3-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-βAla-Peg3-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-βAla-Peg3-Peg3-Peg3,[21-carboxy-heneicosanoyl]-βAla-Peg3-Peg3-Peg3,

[13-carboxy-tridecanoyl]-isoLys-Peg3-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-isoLys-Peg3-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-isoLys-Peg3-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-isoLys-Peg3-Peg3-Peg3,[21-carboxy-heneicosanoyl]-isoLys-Peg3-Peg3-Peg3,

[13-carboxy-tridecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,[15-carboxy-Pentadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,[17-carboxy-Heptadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,[19-carboxy-Nonadecanoyl]-[4-am inobutanoyl]-Peg3-Peg3-Peg3,[21-carboxy-heneicosanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,

[13-carboxy-tridecanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 406),[15-carboxy-Pentadecanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 407),[17-carboxy-Heptadecanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 408),[19-carboxy-Nonadecanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 409),[21-carboxy-heneicosanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 410),

[13-carboxy-tridecanoyl]-isoGlu-KEK-Peg3 (SEQ ID NO: 411),[15-carboxy-Pentadecanoyl]-isoGlu-KEK-Peg3 (SEQ ID NO: 412),[17-carboxy-Heptadecanoyl]-isoGlu-KEK-Peg3 (SEQ ID NO: 413),[19-carboxy-Nonadecanoyl]-isoGlu-KEK-Peg3 (SEQ ID NO: 414),[21-carboxy-heneicosanoyl]-isoGlu-KEK-Peg3 (SEQ ID NO: 415),

[13-carboxy-tridecanoyl]-[4-aminobutanoyl]-KEK-Peg3 (SEQ ID NO: 416),[15-carboxy-Pentadecanoyl]-[4-aminobutanoyl]-KEK-Peg3 (SEQ ID NO: 417),[17-carboxy-Heptadecanoyl]-[4-aminobutanoyl]-KEK-Peg3 (SEQ ID NO: 418),[19-carboxy-Nonadecanoyl]-[4-aminobutanoyl]-KEK-Peg3 (SEQ ID NO: 419),[21-carboxy-heneicosanoyl]-[4-am inobutanoyl]-KEK-Peg3 (SEQ ID NO: 420),

[13-carboxy-tridecanoyl]-isoLys-KEK-Peg3 (SEQ ID NO: 421),[15-carboxy-Pentadecanoyl]-isoLys-KEK-Peg3 (SEQ ID NO: 422),[17-carboxy-Heptadecanoyl]-isoLys-KEK-Peg3 (SEQ ID NO: 423),[19-carboxy-Nonadecanoyl]-isoLys-KEK-Peg3 (SEQ ID NO: 424),[21-carboxy-heneicosanoyl]-isoLys-KEK-Peg3 (SEQ ID NO: 425),

[13-carboxy-tridecanoyl]-βAla-KEK-Peg3 (SEQ ID NO: 426),[15-carboxy-Pentadecanoyl]-βAla-KEK-Peg3 (SEQ ID NO: 427),[17-carboxy-Heptadecanoyl]-βAla-KEK-Peg3 (SEQ ID NO: 428),[19-carboxy-Nonadecanoyl]-βAla-KEK-Peg3 (SEQ ID NO: 429),[21-carboxy-heneicosanoyl]-βAla-KEK-Peg3 (SEQ ID NO: 430),

[13-carboxy-tridecanoyl]-isoGlu-KEK-Peg3-Peg3 (SEQ ID NO: 431),[15-carboxy-Pentadecanoyl]-isoGlu-KEK-Peg3-Peg3 (SEQ ID NO: 432),[17-carboxy-Heptadecanoyl]-isoGlu-KEK-Peg3-Peg3 (SEQ ID NO: 433),[19-carboxy-Nonadecanoyl]-isoGlu-KEK-Peg3-Peg3 (SEQ ID NO: 434),[21-carboxy-heneicosanoyl]-isoGlu-KEK-Peg3-Peg3 (SEQ ID NO: 435),

[13-carboxy-tridecanoyl]-βAla-KEK-Peg3-Peg3 (SEQ ID NO: 436),[15-carboxy-Pentadecanoyl]-βAla-KEK-Peg3-Peg3 (SEQ ID NO: 437),[17-carboxy-Heptadecanoyl]-βAla-KEK-Peg3-Peg3 (SEQ ID NO: 438),[19-carboxy-Nonadecanoyl]-βAla-KEK-Peg3-Peg3 (SEQ ID NO: 439),[21-carboxy-heneicosanoyl]-βAla-KEK-Peg3-Peg3 (SEQ ID NO: 440),

[13-carboxy-tridecanoyl]-isoLys-KEK-Peg3-Peg3 (SEQ ID NO: 441),[15-carboxy-Pentadecanoyl]-isoLys-KEK-Peg3-Peg3 (SEQ ID NO: 442),[17-carboxy-Heptadecanoyl]-isoLys-KEK-Peg3-Peg3 (SEQ ID NO: 443),[19-carboxy-Nonadecanoyl]-isoLys-KEK-Peg3-Peg3 (SEQ ID NO: 444),[21-carboxy-heneicosanoyl]-isoLys-KEK-Peg3-Peg3 (SEQ ID NO: 445),

[13-carboxy-tridecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3 (SEQ ID NO:446), [15-carboxy-Pentadecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3 (SEQ IDNO: 447), [17-carboxy-Heptadecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3(SEQ ID NO: 448),[19-carboxy-Nonadecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3 (SEQ ID NO:449), [21-carboxy-heneicosanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3 (SEQ IDNO: 450),

[13-carboxy-tridecanoyl]-isoGlu-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 451),[15-carboxy-Pentadecanoyl]-isoGlu-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 452),[17-carboxy-Heptadecanoyl]-isoGlu-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 453),[19-carboxy-Nonadecanoyl]-isoGlu-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 454),[21-carboxy-heneicosanoyl]-isoGlu-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 455),

[13-carboxy-tridecanoyl]-βAla-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 456),[15-carboxy-Pentadecanoyl]-βAla-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 457),[17-carboxy-Heptadecanoyl]-βAla-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 458),[19-carboxy-Nonadecanoyl]-βAla-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 459),[21-carboxy-heneicosanoyl]-βAla-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 460),

[13-carboxy-tridecanoyl]-isoLys-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 461),[15-carboxy-Pentadecanoyl]-isoLys-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 462),[17-carboxy-Heptadecanoyl]-isoLys-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 463),[19-carboxy-Nonadecanoyl]-isoLys-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 464),[21-carboxy-heneicosanoyl]-isoLys-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 465),

[13-carboxy-tridecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ IDNO: 466),[15-carboxy-Pentadecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ IDNO: 467),[17-carboxy-Heptadecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ IDNO: 468), [19-carboxy-Nonadecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3-Peg3(SEQ ID NO: 469), [21-carboxy-heneicosanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 470),

[13-carboxy-tridecanoyl]-KEK-isoGlu-Peg3 (SEQ ID NO: 471),[15-carboxy-Pentadecanoyl]-KEK-isoGlu-Peg3 (SEQ ID NO: 472),[17-carboxy-Heptadecanoyl]-KEK-isoGlu-Peg3 (SEQ ID NO: 473),[19-carboxy-Nonadecanoyl]-KEK-isoGlu-Peg3 (SEQ ID NO: 474),[21-carboxy-heneicosanoyl]-KEK-isoGlu-Peg3 (SEQ ID NO: 475),

[13-carboxy-tridecanoyl]-KEK-βAla-Peg3 (SEQ ID NO: 476),[15-carboxy-Pentadecanoyl]-KEK-βAla-Peg3 (SEQ ID NO: 477),[17-carboxy-Heptadecanoyl]-KEK-βAla-Peg3 (SEQ ID NO: 478),[19-carboxy-Nonadecanoyl]-KEK-βAla-Peg3 (SEQ ID NO: 479),[21-carboxy-heneicosanoyl]-KEK-βAla-Peg3 (SEQ ID NO: 480),

[13-carboxy-tridecanoyl]-KEK-[4-aminobutanoyl]-Peg3 (SEQ ID NO: 481),[15-carboxy-Pentadecanoyl]-KEK-[4-aminobutanoyl]-Peg3 (SEQ ID NO: 482),[17-carboxy-Heptadecanoyl]-KEK-[4-aminobutanoyl]-Peg3 (SEQ ID NO: 483),[19-carboxy-Nonadecanoyl]-KEK-[4-aminobutanoyl]-Peg3 (SEQ ID NO: 484),[21-carboxy-heneicosanoyl]-KEK-[4-aminobutanoyl]-Peg3 (SEQ ID NO: 485),

[13-carboxy-tridecanoyl]-KEK-isoLys-Peg3 (SEQ ID NO: 486),[15-carboxy-Pentadecanoyl]-KEK-isoLys-Peg3 (SEQ ID NO: 487),[17-carboxy-Heptadecanoyl]-KEK-isoLys-Peg3 (SEQ ID NO: 488),[19-carboxy-Nonadecanoyl]-KEK-isoLys-Peg3 (SEQ ID NO: 489),[21-carboxy-heneicosanoyl]-KEK-isoLys-Peg3 (SEQ ID NO: 490),

[13-carboxy-tridecanoyl]-KEK-isoGlu-Peg3-Peg3 (SEQ ID NO: 491),[15-carboxy-Pentadecanoyl]-KEK-isoGlu-Peg3-Peg3 (SEQ ID NO: 492),[17-carboxy-Heptadecanoyl]-KEK-isoGlu-Peg3-Peg3 (SEQ ID NO: 493),[19-carboxy-Nonadecanoyl]-KEK-isoGlu-Peg3-Peg3 (SEQ ID NO: 494),[21-carboxy-heneicosanoyl]-KEK-isoGlu-Peg3-Peg3 (SEQ ID NO: 495),

[13-carboxy-tridecanoyl]-KEK-βAla-Peg3-Peg3 (SEQ ID NO: 496),[15-carboxy-Pentadecanoyl]-KEK-βAla-Peg3-Peg3 (SEQ ID NO: 497),[17-carboxy-Heptadecanoyl]-KEK-βAla-Peg3-Peg3 (SEQ ID NO: 498),[19-carboxy-Nonadecanoyl]-KEK-βAla-Peg3-Peg3 (SEQ ID NO: 499),[21-carboxy-heneicosanoyl]-βAla-KEK-Peg3-Peg3 (SEQ ID NO: 500),

[13-carboxy-tridecanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3 (SEQ ID NO:501), [15-carboxy-Pentadecanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3 (SEQ IDNO: 502), [17-carboxy-Heptadecanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3(SEQ ID NO: 503),[19-carboxy-Nonadecanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3 (SEQ ID NO:504), [21-carboxy-heneicosanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3 (SEQ IDNO: 505),

[13-carboxy-tridecanoyl]-KEK-isoLys-Peg3-Peg3 (SEQ ID NO: 506),[15-carboxy-Pentadecanoyl]-KEK-isoLys-Peg3-Peg3 (SEQ ID NO: 507),[17-carboxy-Heptadecanoyl]-KEK-isoLys-Peg3-Peg3 (SEQ ID NO: 508),[19-carboxy-Nonadecanoyl]-KEK-isoLys-Peg3-Peg3 (SEQ ID NO: 509),[21-carboxy-heneicosanoyl]-KEK-isoLys-Peg3-Peg3 (SEQ ID NO: 510),

[13-carboxy-tridecanoyl]-KEK-isoGlu-Peg3-Peg3-Peg3 (SEQ ID NO: 511),[15-carboxy-Pentadecanoyl]-KEK-isoGlu-Peg3-Peg3-Peg3 (SEQ ID NO: 512),[17-carboxy-Heptadecanoyl]-KEK-isoGlu-Peg3-Peg3-Peg3 (SEQ ID NO: 513),[19-carboxy-Nonadecanoyl]-KEK-isoGlu-Peg3-Peg3-Peg3 (SEQ ID NO: 514),[21-carboxy-heneicosanoyl]-KEK-isoGlu-Peg3-Peg3-Peg3 (SEQ ID NO: 515),

[13-carboxy-tridecanoyl]-KEK-βAla-Peg3-Peg3-Peg3 (SEQ ID NO: 516),[15-carboxy-Pentadecanoyl]-KEK-βAla-Peg3-Peg3-Peg3 (SEQ ID NO: 517),[17-carboxy-Heptadecanoyl]-[3Ala-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 518),[19-carboxy-Nonadecanoyl]-KEK-βAla-Peg3-Peg3-Peg3 (SEQ ID NO: 519),[21-carboxy-heneicosanoyl]-KEK-βAla-Peg3-Peg3-Peg3 (SEQ ID NO: 520),

[13-carboxy-tridecanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3-Peg3 (SEQ IDNO: 521),[15-carboxy-Pentadecanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3-Peg3 (SEQ IDNO: 522),[17-carboxy-Heptadecanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3-Peg3 (SEQ IDNO: 523), [19-carboxy-Nonadecanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3-Peg3(SEQ ID NO: 524),[21-carboxy-heneicosanoyl]-KEK-[4-aminobutanoyl]-Peg3-Peg3-Peg3 (SEQ IDNO: 525),

[13-carboxy-tridecanoyl]-KEK-isoLys-Peg3-Peg3-Peg3 (SEQ ID NO: 526),[15-carboxy-Pentadecanoyl]-KEK-isoLys-Peg3-Peg3-Peg3 (SEQ ID NO: 527),[17-carboxy-Heptadecanoyl]-KEK-isoLys-Peg3-Peg3-Peg3 (SEQ ID NO: 528),[19-carboxy-Nonadecanoyl]-KEK-isoLys-Peg3-Peg3-Peg3 (SEQ ID NO: 529),[21-carboxy-heneicosanoyl]-KEK-isoLys-Peg3-Peg3-Peg3 (SEQ ID NO: 530).

Certain preferred substituents Z¹— and Z¹—Z²— include:

[Hexadecanoyl], [Octadecanoyl], [17-Carboxy-heptadecanoyl],[19-Carboxy-nonadecanoyl],

[Hexadecanoyl]-isoGlu, [Octadecanoyl]-isoGlu,

[Hexadecanoyl]-βAla, [Octadecanoyl]-βAla,

[Hexadecanoyl]-isoGlu-Peg3,

[Hexadecanoyl]-βAla-Peg3,

[Hexadecanoyl]-isoGlu-Peg3-Peg3,

[Hexadecanoyl]-βAla-Peg3-Peg3,

[Hexadecanoyl]-βAla-Peg3-Peg3-Peg3,

[Hexadecanoyl]-isoLys,

[Hexadecanoyl]-[4-aminobutanoyl],

[Hexadecanoyl]-isoLys-Peg3,

[Hexadecanoyl]-[4-aminobutanoyl]-Peg3,

[Hexadecanoyl]-isoLys-Peg3-Peg3,

[Hexadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3,

[Hexadecanoyl]-isoLys-Peg3-Peg3-Peg3,

[Hexadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,

[17-carboxy-Heptadecanoyl]-isoGlu,

[19-carboxy-Nonadecanoyl]-isoGlu,

[17-carboxy-Heptadecanoyl]-βAla,

[19-carboxy-Nonadecanoyl]-βAla,

[17-carboxy-Heptadecanoyl]-isoGlu-Peg3,

[19-carboxy-Nonadecanoyl]-isoGlu-Peg3,

[17-carboxy-Heptadecanoyl]-βAla-Peg3,

[19-carboxy-Nonadecanoyl]-βAla-Peg3,

[17-carboxy-Heptadecanoyl]-isoGlu-Peg3-Peg3,

[19-carboxy-Nonadecanoyl]-isoGlu-Peg3-Peg3,

[17-carboxy-Heptadecanoyl]-βAla-Peg3-Peg3,

[19-carboxy-Nonadecanoyl]-βAla-Peg3-Peg3,

[17-carboxy-Heptadecanoyl]-isoGlu-Peg3-Peg3-Peg3,

[19-carboxy-Nonadecanoyl]-isoGlu-Peg3-Peg3-Peg3,

[17-carboxy-Heptadecanoyl]-βAla-Peg3-Peg3-Peg3,

[19-carboxy-Nonadecanoyl]-βAla-Peg3-Peg3-Peg3,

[17-carboxy-Heptadecanoyl]-isoLys,

[19-carboxy-Nonadecanoyl]-isoLys,

[17-carboxy-Heptadecanoyl]-[4-aminobutanoyl],

[19-carboxy-Nonadecanoyl]-[4-aminobutanoyl],

[17-carboxy-Heptadecanoyl]-isoLys-Peg3,

[19-carboxy-Nonadecanoyl]-isoLys-Peg3,

[17-carboxy-Heptadecanoyl]-[4-aminobutanoyl]-Peg3,

[19-carboxy-Nonadecanoyl]-[4-aminobutanoyl]-Peg3,

[17-carboxy-Heptadecanoyl]-isoLys-Peg3-Peg3,

[19-carboxy-Nonadecanoyl]-isoLys-Peg3-Peg3,

[17-carboxy-Heptadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3,

[19-carboxy-Nonadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3,

[17-carboxy-Heptadecanoyl]-isoLys-Peg3-Peg3-Peg3,

[19-carboxy-Nonadecanoyl]-isoLys-Peg3-Peg3-Peg3,

[17-carboxy-Heptadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3,

[19-carboxy-Nonadecanoyl]-[4-aminobutanoyl]-Peg3-Peg3-Peg3.

More preferred substituents Z¹—Z²— include:

[Hexadecanoyl]-isoGlu,

[Hexadecanoyl]-βAla,

[Hexadecanoyl]-isoGlu-Peg3,

[Hexadecanoyl]-βAla-Peg3,

[Hexadecanoyl]-isoGlu-Peg3-Peg3,

[Hexadecanoyl]-isoLys,

[Hexadecanoyl]-isoLys-Peg3,

[Hexadecanoyl]-isoLys-Peg3-Peg3,

[17-carboxy-Heptadecanoyl]-isoGlu,

[19-carboxy-Nonadecanoyl]-isoGlu,

[17-carboxy-Heptadecanoyl]-isoGlu-Peg3,

[19-carboxy-Nonadecanoyl]-isoGlu-Peg3,

[17-carboxy-Heptadecanoyl]-isoGlu-Peg3-Peg3,

[19-carboxy-Nonadecanoyl]-isoGlu-Peg3-Peg3,

[17-carboxy-Heptadecanoyl]-isoGlu-Peg3-Peg3-Peg3,

[19-carboxy-Nonadecanoyl]-isoGlu-Peg3-Peg3-Peg3,

[17-carboxy-Heptadecanoyl]-isoLys,

[19-carboxy-Nonadecanoyl]-isoLys,

[17-carboxy-Heptadecanoyl]-isoLys-Peg3,

[19-carboxy-Nonadecanoyl]-isoLys-Peg3,

[17-carboxy-Heptadecanoyl]-isoLys-Peg3-Peg3,

[19-carboxy-Nonadecanoyl]-isoLys-Peg3-Peg3,

[17-carboxy-Heptadecanoyl]-isoLys-Peg3-Peg3-Peg3,

[19-carboxy-Nonadecanoyl]-isoLys-Peg3-Peg3-Peg3.

Yet further preferred substituents Z¹—Z²— include:

[Hexadecanoyl]-KEK (SEQ ID NO: 251), [Octadecanoyl]-KEK (SEQ ID NO:252),

[Hexadecanoyl]-βAla-Peg3,

[Hexadecanoyl]-KEK-Peg3 (SEQ ID NO: 258),

[Hexadecanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 263),

[Hexadecanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 268),

[17-carboxy-Heptadecanoyl]-KEK (SEQ ID NO: 391),

[19-carboxy-Nonadecanoyl]-KEK (SEQ ID NO: 392),

[17-carboxy-Heptadecanoyl]-KEK-Peg3 (SEQ ID NO: 398),

[19-carboxy-Nonadecanoyl]-KEK-Peg3 (SEQ ID NO: 399),

[17-carboxy-Heptadecanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 403),

[19-carboxy-Nonadecanoyl]-KEK-Peg3-Peg3 (SEQ ID NO: 404),

[17-carboxy-Heptadecanoyl]-isoGlu-KEK (SEQ ID NO: 531),

[19-carboxy-Nonadecanoyl]-isoGlu-KEK (SEQ ID NO: 532),

[17-carboxy-Heptadecanoyl]-isoLys-KEK (SEQ ID NO: 533),

[19-carboxy-Nonadecanoyl]-isoLys-KEK (SEQ ID NO: 534),

[17-carboxy-Heptadecanoyl]-βAla-KEK (SEQ ID NO: 535),

[19-carboxy-Nonadecanoyl]-βAla-KEK (SEQ ID NO: 536),

[17-carboxy-Heptadecanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 408),

[19-carboxy-Nonadecanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ ID NO: 409),

[17-carboxy-Heptadecanoyl]-[4-aminobutanoyl]-KEK (SEQ ID NO: 537),

[19-carboxy-Nonadecanoyl]-[4-aminobutanoyl]-KEK (SEQ ID NO: 538),

[17-carboxy-Heptadecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ IDNO: 468),

[19-carboxy-Nonadecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3-Peg3 (SEQ IDNO: 469),

[17-carboxy-Heptadecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3 (SEQ ID NO:448),

[19-carboxy-Nonadecanoyl]-[4-aminobutanoyl]-KEK-Peg3-Peg3 (SEQ ID NO:449)

[Hexadecanoyl]-isoGlu-KEK-Peg3 (SEQ ID NO: 273),

[Hexadecanoyl]-isoGlu-KEK-Peg3-Peg3 (SEQ ID NO: 293),

[17-carboxy-Heptadecanoyl]-isoGlu-KEK-Peg3-Peg3 (SEQ ID NO: 433),

[19-carboxy-Nonadecanoyl]-isoGlu-KEK-Peg3-Peg3 (SEQ ID NO: 434).

Examples of ψ comprising different substituents (fatty acids, FA),conjugated to the amino acid side-chain, optionally by a spacer, areillustrated below:

Furthermore, the substituent [Hexadecanoyl]-isoGlu, conjugated to theside chain of a lysine residue, is illustrated below:

Thus, the side chain of the Lys residue is covalently attached to theside-chain carboxyl group of the isoGlu spacer —Z2-(—Z^(S1)—) via anamide linkage. A hexadecanoyl group (Z¹) is covalently attached to theamino group of the isoGlu spacer via an amide linkage.

The substituent [Hexadecanoyl]-[4-aminobutanoyl]- conjugated to the sidechain of a lysine residue, is illustrated below

The substituent [(Hexadecanoyl)iso-Lys]- conjugated to the side chain ofa lysine residue, is illustrated below

The substituent [(Hexadecanoyl)β-Ala]- conjugated to the side chain of alysine residue, is illustrated below

Some further specific examples of —Z²—Z¹ combinations are illustratedbelow. In each case, —indicates the point of attachment to the sidechain of the amino acid component of IP:

The skilled person will be well-aware of suitable techniques forpreparing the substituents employed in the context of the invention andconjugating them to the side chain of the appropriate amino acid in thedual agonist peptide. For examples of suitable chemistry, seeWO98/08871, WO00/55184, WO00/55119, Madsen et al., J. Med. Chem.50:6126-32 (2007), and Knudsen et al., J. Med Chem. 43:1664-1669 (2000),incorporated herein by reference.

Synthesis of Dual Agonists

It is preferred to synthesize dual agonists of the invention by means ofsolid-phase or liquid-phase peptide synthesis methodology. In thiscontext, reference may be made to WO 98/11125 and, among many others,Fields, G. B. et al., 2002, “Principles and practice of solid-phasepeptide synthesis”. In: Synthetic Peptides (2nd Edition), and theExamples herein.

In accordance with the present invention, a dual agonist of theinvention may be synthesized or produced in a number of ways, includingfor example, a method which comprises:

(a) synthesizing the dual agonist by means of solid-phase orliquid-phase peptide synthesis methodology and recovering thesynthesized dual agonist thus obtained; or

(b) expressing a precursor peptide sequence from a nucleic acidconstruct that encodes the precursor peptide, recovering the expressionproduct, and modifying the precursor peptide to yield a compound of theinvention.

The precursor peptide may be modified by introduction of one or morenon-proteinogenic amino acids, e.g., Aib, Orn, Dap, or Dab, introductionof a lipophilic substituent Z¹ or Z¹—Z²— at a residue ψ, introduction ofthe appropriate terminal groups R¹ and R², etc.

Expression is typically performed from a nucleic acid encoding theprecursor peptide, which may be performed in a cell or a cell-freeexpression system comprising such a nucleic acid.

It is preferred to synthesize the analogues of the invention by means ofsolid-phase or liquid-phase peptide synthesis. In this context,reference is made to WO 98/11125 and, among many others, Fields, G B etal., 2002, “Principles and practice of solid-phase peptide synthesis”.In: Synthetic Peptides (2nd Edition), and the Examples herein.

For recombinant expression, the nucleic acid fragments encoding theprecursor peptide will normally be inserted in suitable vectors to formcloning or expression vectors. The vectors can, depending on purpose andtype of application, be in the form of plasmids, phages, cosmids,mini-chromosomes, or virus, but also naked DNA which is only expressedtransiently in certain cells is an important vector.

Preferred cloning and expression vectors (plasmid vectors) are capableof autonomous replication, thereby enabling high copy-numbers for thepurposes of high-level expression or high-level replication forsubsequent cloning.

In general outline, an expression vector comprises the followingfeatures in the 5′→3′ direction and in operable linkage: a promoter fordriving expression of the nucleic acid fragment, optionally a nucleicacid sequence encoding a leader peptide enabling secretion (to theextracellular phase or, where applicable, into the periplasma), thenucleic acid fragment encoding the precursor peptide, and optionally anucleic acid sequence encoding a terminator. They may compriseadditional features such as selectable markers and origins ofreplication. When operating with expression vectors in producer strainsor cell lines it may be preferred that the vector is capable ofintegrating into the host cell genome. The skilled person is veryfamiliar with suitable vectors and is able to design one according totheir specific requirements.

The vectors of the invention are used to transform host cells to producethe precursor peptide. Such transformed cells can be cultured cells orcell lines used for propagation of the nucleic acid fragments andvectors, and/or used for recombinant production of the precursorpeptides.

Preferred transformed cells are micro-organisms such as bacteria [suchas the species Escherichia (e.g., E. coli), Bacillus (e.g., Bacillussubtilis), Salmonella, or Mycobacterium (preferably non-pathogenic,e.g., M. bovis BCG), yeasts (e.g., Saccharomyces cerevisiae and Pichiapastoris), and protozoans. Alternatively, the transformed cells may bederived from a multicellular organism, i.e., it may be fungal cell, aninsect cell, an algal cell, a plant cell, or an animal cell such as amammalian cell. For the purposes of cloning and/or optimized expressionit is preferred that the transformed cell is capable of replicating thenucleic acid fragment of the invention. Cells expressing the nucleicfragment can be used for small-scale or large-scale preparation of thepeptides of the invention.

When producing the precursor peptide by means of transformed cells, itis convenient, although far from essential, that the expression productis secreted into the culture medium.

Pharmaceutical Compositions and Administration

An aspect of the present invention relates to a composition comprising adual agonist according to the invention, or a pharmaceuticallyacceptable salt or solvate thereof, together with a carrier. In oneembodiment of the invention, the composition is a pharmaceuticalcomposition and the carrier is a pharmaceutically acceptable carrier.The present invention also relates to a pharmaceutical compositioncomprising a dual agonist according to the invention, or a salt orsolvate thereof, together with a carrier, excipient or vehicle.Accordingly, the dual agonist of the present invention, or salts orsolvates thereof, especially pharmaceutically acceptable salts orsolvates thereof, may be formulated as compositions or pharmaceuticalcompositions prepared for storage or administration, and which comprisea therapeutically effective amount of a dual agonist of the presentinvention, or a salt or solvate thereof.

Suitable salts formed with bases include metal salts, such as alkalimetal or alkaline earth metal salts, for example sodium, potassium ormagnesium salts; ammonia salts and organic amine salts, such as thoseformed with morpholine, thiomorpholine, piperidine, pyrrolidine, a lowermono-, di- or tri-alkylamine (e.g., ethyl-tert-butyl-, diethyl-,diisopropyl-, triethyl-, tributyl- or dimethylpropylamine), or a lowermono-, di- or tri-(hydroxyalkyl)amine (e.g., mono-, di- ortriethanolamine). Internal salts may also be formed. Similarly, when acompound of the present invention contains a basic moiety, salts can beformed using organic or inorganic acids. For example, salts can beformed from the following acids: formic, acetic, propionic, butyric,valeric, caproic, oxalic, lactic, citric, tartaric, succinic, fumaric,maleic, malonic, mandelic, malic, phthalic, hydrochloric, hydrobromic,phosphoric, nitric, sulphuric, benzoic, carbonic, uric,methanesulphonic, naphthalenesulphonic, benzenesulphonic,toluenesulphonic, p-toluenesulphonic (i.e. 4-methylbenzene-sulphonic),camphorsulphonic, 2-aminoethanesulphonic, aminomethylphosphonic andtrifluoromethanesulphonic acid (the latter also being denoted triflicacid), as well as other known pharmaceutically acceptable acids. Aminoacid addition salts can also be formed with amino acids, such as lysine,glycine, or phenylalanine.

In one embodiment, a pharmaceutical composition of the invention is onewherein the dual agonist is in the form of a pharmaceutically acceptableacid addition salt.

As will be apparent to one skilled in the medical art, a“therapeutically effective amount” of a dual agonist compound orpharmaceutical composition thereof of the present invention will varydepending upon, inter alia, the age, weight and/or gender of the subject(patient) to be treated. Other factors that may be of relevance includethe physical characteristics of the specific patient underconsideration, the patient's diet, the nature of any concurrentmedication, the particular compound(s) employed, the particular mode ofadministration, the desired pharmacological effect(s) and the particulartherapeutic indication. Because these factors and their relationship indetermining this amount are well known in the medical arts, thedetermination of therapeutically effective dosage levels, the amountnecessary to achieve the desired result of treating and/or preventingand/or remedying malabsorption and/or low-grade inflammation describedherein, as well as other medical indications disclosed herein, will bewithin the ambit of the skilled person.

As used herein, the term “a therapeutically effective amount” refers toan amount which reduces symptoms of a given condition or pathology, andpreferably which normalizes physiological responses in an individualwith that condition or pathology. Reduction of symptoms or normalizationof physiological responses can be determined using methods routine inthe art and may vary with a given condition or pathology. In one aspect,a therapeutically effective amount of one or more dual agonists, orpharmaceutical compositions thereof, is an amount which restores ameasurable physiological parameter to substantially the same value(preferably to within 30%, more preferably to within 20%, and still morepreferably to within 10% of the value) of the parameter in an individualwithout the condition or pathology in question.

In one embodiment of the invention, administration of a compound orpharmaceutical composition of the present invention is commenced atlower dosage levels, with dosage levels being increased until thedesired effect of preventing/treating the relevant medical indication isachieved. This would define a therapeutically effective amount. For thedual agonists of the present invention, alone or as part of apharmaceutical composition, such human doses of the active dual agonistmay be between about 0.01 μmol/kg and 500 μmol/kg body weight, betweenabout 0.01 μmol/kg and 300 μmol/kg body weight, between 0.01 μmol/kg and100 μmol/kg body weight, between 0.1 μmol/kg and 50 μmol/kg body weight,between 1 μmol/kg and 10 μmol/kg body weight, between 5 μmol/kg and 5μmol/kg body weight, between 10 μmol/kg and 1 μmol/kg body weight,between 50 μmol/kg and 0.1 μmol/kg body weight, between 100 μmol/kg and0.01 μmol/kg body weight, between 0.001 μmol/kg and 0.5 μmol/kg bodyweight, between 0.05 μmol/kg and 0.1 μmol/kg body weight.

The therapeutic dosing and regimen most appropriate for patienttreatment will of course vary with the disease or condition to betreated, and according to the patient's weight and other parameters.Without wishing to be bound by any particular theory, it is expectedthat doses, in the μg/kg range, and shorter or longer duration orfrequency of treatment may produce therapeutically useful results, suchas a statistically significant increase particularly in small bowelmass. In some instances, the therapeutic regimen may include theadministration of maintenance doses appropriate for preventing tissueregression that occurs following cessation of initial treatment. Thedosage sizes and dosing regimen most appropriate for human use may beguided by the results obtained by the present invention, and may beconfirmed in properly designed clinical trials.

An effective dosage and treatment protocol may be determined byconventional means, starting with a low dose in laboratory animals andthen increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Such considerations are known to the skilledperson.

Medical Conditions

In a broad aspect, the present invention provides a dual agonist of theinvention for use as a medicament.

In a further aspect, the present invention relates to a dual agonist ofthe invention for use in therapy.

The dual agonists described in this specification have biologicalactivities of both GLP-1 and GLP-2.

GLP-2 induces significant growth of the small intestinal mucosalepithelium via the stimulation of stem cell proliferation in the cryptsand inhibition of apoptosis on the villi (Drucker et al. Proc Natl AcadSci USA. 1996, 93:7911-6). GLP-2 also has growth effects on the colon.GLP-2 also inhibits gastric emptying and gastric acid secretion(Wojdemann et al. J Clin Endocrinol Metab. 1999, 84:2513-7), enhancesintestinal barrier function (Benjamin et al. Gut. 2000, 47:112-9.),stimulates intestinal hexose transport via the upregulation of glucosetransporters (Cheeseman, Am J Physiol. 1997, R1965-71), and increasesintestinal blood flow (Guan et al. Gastroenterology. 2003, 125, 136-47).

The beneficial effects of GLP-2 in the small intestine have raisedconsiderable interest as to the use of GLP-2 in the treatment ofintestinal disease or injury (Sinclair and Drucker, Physiology 2005:357-65). Furthermore, GLP-2 has been shown to prevent or reduce mucosalepithelial damage in a wide number of preclinical models of gut injury,including chemotherapy-induced enteritis, ischemia-reperfusion injury,dextran sulfate-induced colitis and genetic models of inflammatory boweldisease (Sinclair and Drucker Physiology 2005: 357-65). The GLP-2analogue teduglutide (Gly2-hGLP-2) is approved for treatment of shortbowel syndrome under the trade names GATTEX® and REVESTIVE®.

GLP-1 is a peptide hormone known for its important role in glucosehomeostasis. When secreted from the gastrointestinal tract in responseto nutrient ingestion, GLP-1 potentiates glucose-stimulated insulinsecretion from the β-cells (Kim and Egan, 2008, Pharmacol.Rev. 470-512).Furthermore, GLP-1 or its analogues has been shown to increasesomatostatin secretion and suppress glucagon secretion (Hoist J J, 2007,Physiol Rev. 1409-1439).

Besides the primary actions of GLP-1 on glucose-stimulated insulinsecretion, GLP-1 is also known as a key regulator of appetite, foodintake, and body weight. Moreover, GLP-1 can inhibit gastric emptyingand gastrointestinal motility in both rodents and humans, most likelythrough GLP-1 receptors present in the gastrointestinal tract (Hoist JJ, 2007, Physiol Rev. 1409-1439; Hellstrom et al., 2008,Neurogastroenterol. Motil. June; 20(6):649-659). In addition, GLP-1seems to have insulin-like effects in major extrapancreatic tissues,participating in glucose homeostasis and lipid metabolism in tissuessuch as muscle, liver, and adipose tissues (Kim and Egan, 2008,Pharmacol.Rev. 470-512).

Thus, the dual agonist compounds of the present invention may be used toincrease intestinal mass, improve intestinal function (especiallyintestinal barrier function), increase intestinal blood flow, or repairintestinal damage or dysfunction (whether structural or functional),e.g., damage to the intestinal epithelium. They may also be used in theprophylaxis or treatment of conditions which may be ameliorated by theseeffects, and in reducing the morbidity related to gastrointestinaldamage.

The dual agonists therefore find use in many gastrointestinal disorders.The term “gastrointestinal” is used here to include the entiregastrointestinal tract, including oesophagus, stomach, small intestine(duodenum, jejunum, ileum) and large intestine (cecum, colon, rectum),but especially the small intestine and colon.

Thus, conditions in which the dual agonists may be of benefit includemalabsorption, ulcers (which may be of any etiology, e.g., pepticulcers, Zollinger-Ellison Syndrome, drug-induced ulcers, and ulcersrelated to infections or other pathogens), short-bowel syndrome,cul-de-sac syndrome, inflammatory bowel disease (Crohn's disease andulcerative colitis), irritable bowel syndrome (IBS), pouchitis, celiacsprue (for example arising from gluten induced enteropathy or celiacdisease), tropical sprue, hypogammaglobulinemic sprue, and mucositis ordiarrhea induced by chemotherapy or radiation therapy.

The dual agonists may also find use in certain conditions which do notprimarily affect gastrointestinal tissue but which may be caused orexacerbated by factors arising from intestinal dysfunction. For example,impaired intestinal barrier function (which may be referred to as“leakiness” of the intestine or gut) can lead to transit of materialsfrom the lumen of the gut directly into the bloodstream and thus to thekidney, lung and/or liver. These materials may include food moleculessuch as fats, which contribute to hepatitis and/or fatty liver diseases,including parenteral nutrition associated gut atrophy, PNALD (ParenteralNutrition-Associated Liver Disease), NAFLD (Non-Alcoholic Fatty LiverDisease) and NASH (Non-Alcoholic Steatohepatitis). The materialscrossing into the bloodstream may also include pathogens such asbacteria, and toxins such as bacterial lipopolysaccharide (LPS), whichmay contribute to systemic inflammation (e.g. vascular inflammation).Such inflammation is often referred to as “low grade inflammation” andis a contributing factor to the pathogenesis of metabolic endotoxemia (acondition seen in both diabetes and obesity, discussed further below),primary biliary cirrhosis and hepatitis. Entry of pathogens to thebloodstream may also result in conditions such as necrotisingenterocolitis.

Low grade inflammation is not characterised by the normal symptoms ofacute inflammation such as pain, fever and redness, but can be detectedvia the presence of inflammatory markers in the blood, such asC-reactive protein and pro-inflammatory cytokines including TNF-alpha(tumour necrosis factor alpha).

The dual agonists may also find use in conditions which primarily affectother tissues but have gastrointestinal side-effects. For example,inflammatory conditions such as pancreatitis result in elevated levelsof circulating inflammatory mediators which may in turn induceintestinal damage or intestinal dysfunction, such as impairment ofbarrier function. In some circumstances, this may lead to more severesystemic inflammatory conditions such as sepsis, or to surgicalprocedures or mechanical injuries (volvulus) where blood supply to theintestine is interrupted, ultimately leading to ischaemia-reperfusioninjuries.

Similarly, graft versus host disease (GVHD) may result in substantialtissue damage to the gastrointestinal tract, resulting in impairedbarrier function and other side effects such as diarrhea. Thus, the dualagonists described may be useful for the prophylaxis or treatment ofintestinal dysfunction or damage caused by or associated with GVHD, aswell as prophylaxis or treatment of side effects such as diarrhea causedby or associated with GVHD.

The dual agonist compounds described herein also find use, inter alia,in reducing or inhibiting weight gain, reducing rate of gastric emptyingor intestinal transit, reducing food intake, reducing appetite, orpromoting weight loss. The effect on body weight may be mediated in partor wholly via reducing food intake, appetite or intestinal transit.

Thus, the dual agonists of the invention can be used for the prophylaxisor treatment of obesity, morbid obesity, obesity-linked gallbladderdisease and obesity-induced sleep apnea.

Independently of their effect on body weight, the dual agonists of theinvention may have a beneficial effect on glucose tolerance and/orglucose control. They may also be used to modulate (e.g. improve)circulating cholesterol levels, being capable of lowering circulatingtriglyceride or LDL levels, and increasing HDL/LDL ratio.

Thus, they may be used for the prophylaxis or treatment of inadequateglucose control, glucose tolerance or dyslipidemia (e.g., elevated LDLlevels or reduced HDL/LDL ratio) and associated conditions, includingdiabetes (e.g., Type 2 diabetes, gestational diabetes), pre-diabetes,metabolic syndrome and hypertension.

Many of these conditions are also associated with obesity or overweight.The effects of the dual agonists on these conditions may thereforefollow from their effect on body weight, in whole or in part, or may beindependent thereof.

Effects on body weight may be therapeutic or cosmetic.

The dual agonist activity of the compounds described herein may beparticularly beneficial in many of the conditions described, as the twoactivities may complement one another.

For example, malabsorption is a condition arising from abnormality inthe absorption of water and/or food nutrients, such as amino acids,sugars, fats, vitamins or minerals, via the gastrointestinal (GI) tract,leading to malnutrition and/or dehydration. Malabsorption may be aresult of physical (e.g. traumatic) or chemical damage to the intestinaltract. Dual agonists as described in this specification may be capableof improving intestinal barrier function, reducing gastric emptying, andincreasing intestinal absorption while at the same time normalisingintestinal transit time. This would not only help patients to increasethe absorption of nutrients and liquid, but would also alleviatepatients' social problems related to meal-stimulated bowel movements.

Furthermore, intestinal function and metabolic disorders may be closelyinter-related, with each contributing to the development or symptoms ofthe other.

As mentioned above, obesity is linked with low grade inflammation(sometimes designated “obesity-linked inflammation”). It is alsogenerally recognised that obesity (along with other syndromes) causes anincreased vascular permeability which allows pathogens and toxins suchas LPS to enter the cell wall of the intestinal tract and therebyinitiate inflammation. The changes that result from the inflammatoryresponse are essentially the same regardless of the cause and regardlessof where the insult arises. The inflammatory response may be acute(short lived) or chronic (longer lasting).

It has been demonstrated that, e.g., obese mice (ob/ob and db/db mice)have a disrupted mucosal barrier function and exhibit increasedlow-grade inflammation (Brun et al., 2007, Am. J. Physiol. Gastrointest.Liver Physiol., 292: G518-G525, Epub 5 Oct. 2006). These observationswere further extended to C57BL6/J mice maintained on a high-fat diet(Cani et al., 2008, Diabetes, vol. 57, 1470-1481) and to non-obesediabetic mice (Hadjiyanni et al., 2009, Endocrinology, 150(2): 592-599).

Cani and colleagues (Gut; 2009, 58:1091-1103,) reported that in ob/obmice, the modulation of the gut microbiota resulted in decreasedintestinal barrier dysfunction and reduced systemic inflammation via aGLP-2 dependent pathway. Further, the increased intestinal permeabilityobserved in obese and diabetic patients is likely to play a more vitalrole in the disease progression than previously anticipated. Increasedintestinal permeability leads to increased bacterial lipopolysaccharide(LPS) transport across the intestinal barrier. This increased LPSactivates immune cells, such as circulating macrophages and macrophagesresiding in organs in the body, causing low-grade chronic inflammationthat may be involved in the pathogenesis of many diseases. Thisphenomenon is called metabolic endotoxemia (ME).

The inflammatory process may also play a role in causing metabolicdysfunction in obese individuals, such as insulin resistance and othermetabolic disturbances.

Thus, the dual agonist compounds of the invention may be particularlyuseful for prophylaxis or treatment of low grade inflammation,especially in obese or overweight individuals, exerting beneficialeffects via the GLP-1 agonist component of their activity and/or theGLP-2 component of their activity.

The therapeutic efficacy of treatment with a dual agonist of theinvention may be monitored by enteric biopsy to examine the villusmorphology, by biochemical assessment of nutrient absorption, bynon-invasive determination of intestinal permeability, by patient weightgain, or by amelioration of the symptoms associated with theseconditions.

In a further aspect there is provided a therapeutic kit comprising adual agonist of the invention, or a pharmaceutically acceptable salt orsolvate thereof.

The following examples are provided to illustrate preferred aspects ofthe invention and are not intended to limit the scope of the inventionin any way.

EXAMPLES

The following examples are provided to illustrate preferred aspects ofthe invention and are not intended to limit the scope of the invention.

Materials and Methods

General Peptide Synthesis

List of abbreviations and suppliers are provided in the table below

List of abbreviations and suppliers Abbreviation Name Brand/SupplierResins TENTAGEL ™ PHB AA(Proct)-Fmoc Rapp Polymere TENTAGEL ™ SRAM RappPolymere Amino Pseudoprolines (E.g. QT, AT, FS) Jupiter Bioscience Ltd.acids Fmoc-L-AA-OH Senn Chemicals AG Coupling COMU (1-Cyano-2-ethoxy-2-Watson International Ltd. reagents oxoethylidenaminooxy)dimethyl-amino-morpholino-carbenium hexafluorophosphate DICDiisopropylcarbodiimide Fluka/Sigma Aldrich Co. HATUN-[(dimethylamino)-1 H-1,2,3-tri- ChemPep Inc.azol[4,5-b]pyridine-1-ylmethylene]- N-methylmethanaminiumhexafluorophosphate N-oxide HOBt Hydroxybenzotriazole Sigma-Aldrich Co.Solvents Boc₂O Di-tert-butyl pyrocarbonate Advanced ChemTech reagentsDCM Dichloromethane Prolabo (VWR) DIPEA DiisopropylethylamineFluka/Sigma Aldrich Co. DMF N,N-dimethylformamide Taminco DODT3,6-dioxa-1,8-octanedithiol Sigma-Aldrich Co. Et₂O Diethyl ether Prolabo(VWR) EtOH Ethanol CCS Healthcare AB Formic acid (HPLC) Sigma-AldrichCo. H₂O Water, MILLI-Q ™ water Millipore MeCN Acetonitrile (HPLC)Sigma-Aldrich Co. NMP N-methylpyrrolidone Sigma-Aldrich Co. PiperidineJubliant Life Sciences Ltd. TFA Trifluoroacetic acid (HPLC) ChemicalsRaw Materials Ltd. TIS Triisopropylsilane Sigma-Aldrich Co. MeOHMethanol Sigma-Aldrich Co.

Apparatus and Synthetic Strategy

Peptides were synthesized batchwise on a peptide synthesizer, such as aCEM LIBERTY™ Peptide Synthesizer or a SYMPHONY® X Synthesizer, accordingto solid phase peptide synthetic procedures using9-fluorenylmethyloxycarbonyl (Fmoc) as N-α-amino protecting group andsuitable common protection groups for side-chain functionalities.

As polymeric support-based resins, such as e.g. TENTAGEL™, was used. Thesynthesizer was loaded with resin that prior to usage was swelled inDMF.

Coupling

CEM LIBERTY™ Peptide Synthesizer

A solution of Fmoc-protected amino acid (4 equiv.) was added to theresin together with a coupling reagent solution (4 equiv.) and asolution of base (8 equiv.). The mixture was either heated by themicrowave unit to 70-75° C. and coupled for 5 minutes or coupled with noheat for 60 minutes. During the coupling nitrogen was bubbled throughthe mixture.

SYMPHONY® X Synthesizer

The coupling solutions were transferred to the reaction vessels in thefollowing order: amino acid (4 equiv.), HATU (4 equiv.) and DIPEA (8equiv.). The coupling time was 10 min at room temperature (RT) unlessotherwise stated. The resin was washed with DMF (5×0.5 min). In case ofrepeated couplings the coupling time was in all cases 45 min at RT.

Deprotection

CEM LIBERTY™ Peptide Synthesizer

The Fmoc group was deprotected using piperidine in DMF or other suitablesolvents. The deprotection solution was added to the reaction vessel andthe mixture was heated for 30 sec. reaching approx. 40° C. The reactionvessel was drained and fresh deprotection solution was added andsubsequently heated to 70-75° C. for 3 min. After draining the reactionvessel, the resin was washed with DMF or other suitable solvents.

SYMPHONY® X Synthesizer

Fmoc deprotection was performed for 2.5 minutes using 40% piperidine inDMF and repeated using the same conditions. The resin was washed withDMF (5×0.5 min).

Side-Chain Acylation

A suitable trifunctional amino acid with an orthogonal side chainprotecting group according to Fmoc methodology is introduced at theposition of the acylation. The N-terminal of the growing peptide chainis then Boc-protected using Boc₂O or alternatively by using anN-α-Boc-protected amino acid in the last coupling. While the peptide isstill attached to the resin, the orthogonal side chain protecting groupis selectively cleaved using a suitable deprotection reagent. Thelipophilic moiety is then coupled directly to the free sidechainfunctionality or alternatively via a linker in between according tosuitable coupling protocols.

Cleavage

The dried peptide resin was treated with TFA and suitable scavengers forapproximately 2 hours. The volume of the filtrate was reduced and thecrude peptide was precipitated after addition of diethylether. The crudepeptide precipitate was washed several times with diethylether andfinally dried.

HPLC Purification of the Crude Peptide

The crude peptide was purified by preparative reverse phase HPLC using aconventional HPLC apparatus, such as a Gilson GX-281 with 331/332 pumpcombination, for binary gradient application equipped with a column,such as 5×25 cm Gemini NX 5u C18 110A column, and a fraction collectorusing a flow 20-40 ml/min with a suitable gradient of buffer A (0.1%Fomic acid, aq.) or A (0.1% TFA, aq.) and buffer B (0.1% Formic acid,90% MeCN, aq.) or B (0.1% TFA, 90% MeCN, aq.). Fractions were analyzedby analytical HPLC and MS and selected fractions were pooled andlyophilized. The final product was characterized by HPLC and MS.

Analytical HPLC

Final purities were determined by analytic HPLC (Agilent 1100/1200series) equipped with auto sampler, degasser, 20 μl flow cell andCHROMELEON™ software. The HPLC was operated with a flow of 1.2 ml/min at40° C. using an analytical column, such as Kinetex 2.6 μm XB-C18 100A100×4.6 mm column. The compound was detected and quantified at 215 nm.Buffers A (0.1% TFA, aq.) and buffer B (0.1% TFA, 90% MeCN, aq.).

Mass Spectroscopy

Final MS analysis were determined on a conventional mass spectroscopy,e.g., Waters XEVO® G2 Tof, equipped with electrospray detector withlock-mass calibration and MASSLYNX™ software. It was operated inpositive mode using direct injection and a cone voltage of 15V (1 TOF),30 V (2 TOF) or 45 V (3 TOF) as specified on the chromatogram. Precisionwas 5 ppm with a typical resolution of 15,000-20,000.

GLP-1 and GLP-2 Receptor Efficacy Assays

Peptides of this invention function as both GLP-1 and GLP-2 agonists andthus activate the GLP-1 receptor and GLP-2 receptor, respectively. Oneuseful in vitro assay for measuring GLP-1 and GLP-2 receptor activity isquantitation of cAMP, i.e. 3′-5′-cyclic adenosine monophosphate, whichis a second messenger essential in many biological processes, and one ofthe most ubiquitous mechanisms for regulating cellular functions. Anexample is the cAMP ALPHASCREEN® assay from Perkin Elmer which has beenused to quantitate the cAMP response upon GLP-1 and GLP-2 receptoractivation in HEK293 cells stably expressing GLP-1 R or GLP-2 R. Testcompounds eliciting an increase in the intracellular level of cAMP canbe tested in these assays, and the response normalized relative to apositive and negative control (vehicle) to calculate the EC50 andmaximal response from the concentration response curve using the4-parameter logistic (4PL) nonlinear model for curve fitting.

Example 1: Synthesis of the Compounds

Compounds Synthesised

The following compounds of Table 1 were synthesized using the abovetechniques.

TABLE 1 Compounds synthesized  1Hy-H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]EAARDFIAWLIEHKITD-OH (SEQ ID NO: 183)  2Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]EAARDFIAWLIEHKITD-OH (SEQ ID NO: 184)  3Hy-H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]EAARDFIAWLIEHKITD-OH (SEQ ID NO: 185)  4Hy-H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]KAARDFIAWLIEHKITD-OH (SEQ ID NO: 186)  5Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]KAARDFIAWLIEHKITD-OH (SEQ ID NO: 187)  6Hy-H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]KAARDFIAWLIEHKITD-OH (SEQ ID NO: 188)  7Hy-H[Aib]EGTFSSELATILDG[K([17-carboxy-heptadecanoyl]-isoGlu)]AARDFIAWLIEHKITD-OH (SEQ ID NO: 189)  8Hy-H[Aib]EGSFTSELATILDG[K([17-carboxy-heptadecanoyl]-isoGlu)]AARDFIAWLIEHKITD-OH (SEQ ID NO: 190)  9Hy-H[Aib]EGTFTSELATILDG[K([17-carboxy-heptadecanoyl]-isoGlu)]AARDFIAWLIEHKITD-OH (SEQ ID NO: 191) 10Hy-H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIEHKITD-OH (SEQ ID NO: 192) 11Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIEHKITD-OH (SEQ ID NO: 193) 12Hy-H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIEHKITD-OH (SEQ ID NO: 194) 13Hy-H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIAHKITD-OH (SEQ ID NO: 195) 14Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIAHKITD-OH (SEQ ID NO: 196) 15Hy-H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]LAARDFIAWLIAHKITD-OH (SEQ ID NO: 197) 16Hy-H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIQHKITD-OH (SEQ ID NO: 199) 18Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIQHKITD-OH (SEQ ID NO: 200) 19Hy-H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIQHKITD-OH (SEQ ID NO: 201) 20Hy-H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIEHKITD-OH (SEQ ID NO: 202) 21Hy-H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIAHKITD-OH (SEQ ID NO: 203) 22Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIAHKITD-OH (SEQ ID NO: 204) 23Hy-H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIAHKITD-OH (SEQ ID NO: 205) 24Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIEHKITD-OH (SEQ ID NO: 206) 25Hy-H[Aib]EGTFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIEHKITD-OH (SEQ ID NO: 207) 26Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIHHKITD-OH (SEQ ID NO: 208) 27Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIYHKITD-OH (SEQ ID NO: 209) 28Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLILHKITD-OH (SEQ ID NO: 210) 29Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIKHKITD-OH (SEQ ID NO: 211) 30Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyq-isoGlu)]QAARDFIAWLIRHKITD-OH (SEQ ID NO: 212) 31Hy-H[Aib]EGSFTSELATILD[K([17-Carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLISHKITD-OH (SEQ ID NO: 213) 32Hy-H[Aib]EGSFTSELATILD[K([Hexadecanoyl]-βAla)]QAARDFIAWLQQHKITD-OH (SEQ ID NO: 214) 33Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu-Peg3)+QAARDFIAWLYQHKITD-OH (SEQ ID NO: 215) 34Hy-H[Aib]EGSFTSELATILD[K([19-carboxy-nonadecanoyl]-isoGlu-Peg3-Peg3)+QAARDFIAWLKQHKITD-OH (SEQ ID NO: 216) 35Hy-H[Aib]EGSFTSELATILD[K([19-carboxy-nonadecanoyl]iso-Lys-Peg3-Peg3-Peg3)+QAARDFIAWLIQQKITD-OH (SEQ ID NO: 217) 36Hy-H[Aib]EGSFTSELATILD[K(Octadecanoyl)]QAARDFIAWLIQYKITD-OH (SEQID NO: 218) 37Hy-H[Aib]EGTFSSELSTILE[K(Hexadecanoyl-isoGlu)]QASREFIAWLIAYKITE-OH (SEQ ID NO: 219) 38Hy-H[Aib]EGTFSSELATILDEQAARDFIAWLIAHKITDkkkkkk([17-carboxy-Heptadecanoyl]-isoGlu)]-[NH2] (SEQ ID NO: 220) 39Hy-H[Aib]EGTFTSELATILDEQAARDFIAWLIAHKITDkkkkkk([17-carboxy-Heptadecanoyl]-isoGlu)]-[NH2] (SEQ ID NO: 221) 40Hy-H[Aib]EGSFTSELATILDEQAARDFIAWLIEHKITDkkkkkk([17-carboxy-Heptadecanoyl]-isoGlu)]-[NH2] (SEQ ID NO: 222) 41Hy-H[Aib]EGTFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu)]QAARDFIAWLIQHKITD-OH (SEQ ID NO: 223) 42Hy-H[Aib]EGSFTSE[K([19-carboxy-nonadecanoyl]-isoGlu-Peg3-Peg3)]ATILDEQAARDFIAWLIEHKITD-OH (SEQ ID NO: 224) 43Hy-H[Aib]EGSFTSELATILD[K([19-carboxy-nonadecanoyl]-isoGlu-Peg3-Peg3)]KAARDFIAWLIEHKITD-OH (SEQ ID NO: 225) 44Hy-H[Aib]EGSFTSELATILEG[K([19-carboxy-nonadecanoyl]-isoGlu-Peg3-Peg3)]AARDFIAWLIEHKITD-OH (SEQ ID NO: 226) 45Hy-H[Aib]EGSFTSELATILDEQAA[K([19-carboxy-nonadecanoyl]-isoGlu-Peg3-Peg3)(SEQ ID NO: 227) 46Hy-H[Aib]EGTFTSELATILDEQAA[K([19-carboxy-nonadecanoyl]-isoGlu-Peg3-Peg3)(SEQ ID NO: 228) 47Hy-H[Aib]EGTFSSELATILD[K([17-Carboxy-heptadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIQHKITD-OH (SEQ ID NO: 229) 48Hy-H[Aib]EGTFSSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIQHKITD-OH (SEQ ID NO: 230) 49Hy-H[Aib]EGTFSSELATILD[K([17-Carboxy-heptadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIEHKITD-OH (SEQ ID NO: 231) 50Hy-H[Aib]EGTFSSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIEHKITD-OH (SEQ ID NO: 232) 51Hy-H[Aib]EGTFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK)]QAARDFIAWLIQHKITD-OH (SEQ ID NO: 233) 52Hy-H[Aib]EGTFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIQHKITD-OH (SEQ ID NO: 234) 53Hy-H[Aib]EGSFTSE[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]ATILDEQAARDFIAWLIEHKITD-OH (SEQ ID NO: 235) 54Hy-H[Aib]EGTFTSE[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]ATILDEQAARDFIAWLIEHKITD-OH (SEQ ID NO: 236) 55Hy-H[Aib]EGSFTSE[K([19-carboxy-nonadecanoyl]iso-Glu-KEK-Peg3-Peg3)]ATILDEQAARDFIAWLIEHKITD-OH (SEQ ID NO: 237) 56Hy-H[Aib]EGTFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIEHKITD-OH (SEQ ID NO: 238) 57Hy-H[Aib]EGSFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIEHKITD-OH (SEQ ID NO: 239) 58Hy-H[Aib]EGSFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]QAARDFIAWLIAHKITD-OH (SEQ ID NO: 240) 59Hy-H[Aib]EGSFTSELATILD[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]KAARDFIAWLIEHKITD-OH (SEQ ID NO: 241) 60Hy-H[Aib]EGSFTSELATILD[K([19-carboxy-nonadecanoyl]iso-Glu-KEK-Peg3-Peg3)]QAARDFIAWLIEHKITD-OH (SEQ ID NO: 242) 61Hy-H[Aib]EGSFTSELATILEG[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]AARDFIAWLIEHKITD-OH (SEQ ID NO: 243) 62Hy-H[Aib]EGSFTSELATILDA[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)]AARDFIAWLIEHKITD-OH (SEQ ID NO: 244) 63Hy-H[Aib]EGSFTSELATILDA[K([19-carboxy-nonadecanoyl]iso-Glu-KEK-Peg3-Peg3)]AARDFIAWLIEHKITD-OH (SEQ ID NO: 245) 64Hy-H[Aib]EGSFTSELATILDEQAA[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)(SEQ ID NO: 246) 65Hy-H[Aib]EGTFTSELATILDEQAA[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)(SEQ ID NO: 247) 66Hy-H[Aib]EGSFTSELATILDEQAA[K([19-carboxy-nonadecanoyl]iso-Glu-KEK-Peg3-Peg3)(SEQ ID NO: 248) 67Hy-H[Aib]EGTFTSELATILDEQAA[K([19-carboxy-nonadecanoyl]iso-Glu-KEK-Peg3-Peg3)(SEQ ID NO: 249) 68Hy-H[Aib]EGSFTSELATILDAKAA[K([19-Carboxy-nonadecanoyl]-isoGlu-KEK-Peg3)(SEQ ID NO: 250)

The following reference compounds A and B were also synthesized:

A (SEQ ID NO: 539) Hy-H[Aib]DGSFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIQHKITD-OH B (SEQ ID NO: 540)Hy-H[Aib]EGSFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIQHKITD-OH

For illustration purposes only, the synthesis of two selected compoundsis described in detail below.

Synthesis of Compound 17

(SEQ ID NO: 199) H-H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIQHKITD-OH

Solid phase peptide synthesis was performed on a SYMPHONY® X Synthesizerusing standard Fmoc chemistry. TENTAGEL™ S PHB Asp(tBu)Fmoc (1.15 g;0.23 mmol/g) was swelled in DMF (10 ml) prior to use and the Fmoc-groupwas deprotected according to the procedure described above.

Coupling

Suitable protected Fmoc-amino acids according to the sequence werecoupled as described above using HATU as coupling reagent. All couplingswere performed at R.T. In order to facilitate the synthesis, apseudoproline were used: in position 6 and 7 Fmoc-Phe-Ser(psiMe,Mepro)-OH. Acylation in position 16 was obtained according to theside-chain acylation procedure described above. The pseudoproline wascoupled according to the standard procedure described above forFmoc-amino acids.

Deprotection

Fmoc deprotection was performed according to the procedure describedabove.

Cleavage of the Peptide from the Solid Support

The peptide-resin was washed with EtOH (3×10 ml) and Et2O (3×10 ml) anddried to constant weight at room temperature (r.t.). The peptide wascleaved from the resin by treatment with TFA/TIS/H₂O (95/2.5/2.5; 40 ml,2 h; r.t.). The volume of the filtrate was reduced and the crude peptidewas precipitated after addition of diethylether. The crude peptideprecipitate was washed several times with diethylether and finally driedto constant weight at room temperature yield 1100 mg crude peptideproduct (purity ˜40%).

HPLC Purification of the Crude Peptide

The crude peptide was purified by preparative reverse phase HPLC using aGilson GX-281with 331/332 pump combination for binary gradientapplication equipped with a 5×25 cm Gemini NX 5u C18 110A, column and afraction collector and run at 35 ml/min with a gradient of buffer A(0.1% TFA, aq.) and buffer B (0.1% TFA, 90% MeCN, aq.) gradient from 25%B to 60% B in 47 min. Fractions were analyzed by analytical HPLC and MSand relevant fractions were pooled and lyophilized to yield 105.7 mg,with a purity of 91% as characterized by HPLC and MS as described above.Calculated monoisotopic MW=4164.21, found 4164.23.

Synthesis of Compound 4

(SEQ ID NO: 186) H-H[Aib]EGTFSSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]KAARDFIAWLIEHKITD-OH

Solid phase peptide synthesis was performed on a SYMPHONY® X Synthesizerusing standard Fmoc chemistry. TENTAGEL™ S PHB Asp(tBu)Fmoc (1.20 g;0.23 mmol/g) was swelled in DMF (10 ml) prior to use and the Fmoc-groupwas deprotected according to the procedure described above.

Coupling

Suitable protected Fmoc-amino acids according to the sequence werecoupled as described above using HATU as coupling reagent. All couplingswere performed at R.T. In order to facilitate the synthesis, apseudoproline were used: in position 6 and 7 Fmoc-Phe-Ser(psiMe,Mepro)-OH. Acylation in position 16 was obtained according to theside-chain acylation procedure described above. The pseudoproline wascoupled according to the standard procedure described above forFmoc-amino acids.

Deprotection

Fmoc deprotection was performed according to the procedure describedabove.

Cleavage of the Peptide from the Solid Support

The peptide-resin was washed with EtOH (3×10 ml) and Et2O (3×10 ml) anddried to constant weight at room temperature (r.t.). The peptide wascleaved from the resin by treatment with TFA/TIS/H₂O (95/2.5/2.5; 40 ml,2 h; r.t.). The volume of the filtrate was reduced and the crude peptidewas precipitated after addition of diethylether. The crude peptideprecipitate was washed several times with diethylether and finally driedto constant weight at room temperature yield 900 mg crude peptideproduct (purity ˜35%).

HPLC Purification of the Crude Peptide

The crude peptide was purified by preparative reverse phase HPLC using aGilson GX-281with 331/332 pump combination for binary gradientapplication equipped with a 5×25 cm Gemini NX 5u C18 110A, column and afraction collector and run at 35 ml/min with a gradient of buffer A(0.1% TFA, aq.) and buffer B (0.1% TFA, 90% MeCN, aq.) gradient from 30%B to 65% B in 47 min. Fractions were analyzed by analytical HPLC and MSand relevant fractions were pooled and lyophilized to yield 100,7 mg,with a purity of 91% as characterized by HPLC and MS as described above.Calculated monoisotopic MW=4165.23, found 4165.26.

Example 2: GLP-1R and GLP-2R EC₅₀ Measurements

Generation of cell line expressing human GLP-1 receptors

The cDNA encoding the human glucagon-like peptide 1 receptor (GLP-1 R)(primary accession number P43220) was cloned from the cDNA BC112126(MGC:138331/IMAGE:8327594). The DNA encoding the GLP-1-R was amplifiedby PCR using primers encoding terminal restriction sites for subcloning.The 5′-end primers additionally encoded a near Kozak consensus sequenceto ensure efficient translation. The fidelity of the DNA encoding theGLP-1-R was confirmed by DNA sequencing. The PCR products encoding theGLP-1-R were subcloned into a mammalian expression vector containing aneomycin (G418) resistance marker. The mammalian expression vectorsencoding the GLP-1-R were transfected into HEK293 cells by a standardcalcium phosphate transfection method. 48 hours post-transfection, cellswere seeded for limited dilution cloning and selected with 1 mg/ml G418in the culture medium. Following 3 weeks in G418 selection clones werepicked and tested in a functional GLP-1 receptor potency assay asdescribed below. One clone was selected for use in compound profiling.

Generation of Cell Line Expressing Human GLP-2 Receptors

The hGLP2-R was purchased from MRC-geneservice, Babraham, Cambridge asan Image clone: 5363415 (11924-I17). For subcloning into a mammalianexpression vector, primers for subcloning were obtained fromDNA-Technology, Risskov, Denmark. The 5′ and 3′ primers used for the PCRreaction include terminal restriction sites for cloning and the contextof the 5′ primer is modified to a Kozak consensus without changing thesequence of the product encoded by the ORF. A standard PCR reaction wasrun using Image clone 5363415 (11924-117) as a template with theabove-mentioned primers and Polymerase HERCULASE® II Fusion in a totalvol. of 50μI. The generated PCR product was purified using GFX PCR andGel band purification kit, digested with restriction enzymes and clonedinto the mammalian expression vector using Rapid DNA Ligation Kit.Ligation reaction was transformed to XL10 Gold Ultracompetent cells andcolonies were picked for DNA production using ENDOFREE® Plasmid maxikit. Subsequent sequence analysis was conducted by MWG Eurofins,Germany. The clone was confirmed to be the hGLP-2 (1-33) receptor,splice variant rs17681684.

HEK293 cells were transfected using the LIPOFECTAMINE® PLUS transfectionmethod. The day before transfection, HEK293 cells were seeded in two T75flasks at a density of 2×10⁶ cells/T75 flask in cell culturing mediumwithout antibiotics. On the day of transfection, cells were washed with1× DPBS and medium was replaced with OPTI-MEM® to a volume of 5 mL/T75flask before addition of LIPOFECTAMINE®-plasmid complexes were addedgently and drop wise to the cells in T75 flasks and replaced with growthmedium after 3 hours and again to growth medium supplemented with 500μg/mL G418 after 24 hours. Following 4 weeks in G418 selection, cloneswere picked and tested in a functional GLP-2 receptor potency assay asdescribed below. One clone was selected for use in compound profiling.

GLP-1 R and GLP-2 Receptor Potency Assays

The cAMP ALPHASCREEN® assay from Perkin Elmer was used to quantitate thecAMP response to activation of the GLP1 and GLP2 receptor, respectively.Exendin-4 was used as reference compound for GLP1 receptor activationand Teduglutide as reference compound for GLP2 receptor activation. Datafrom test compounds eliciting an increase in the intracellular level ofcAMP were normalized relative to the positive and negative control(vehicle) to calculate the EC₅₀ and maximal response from theconcentration response curve. The results are listed in Table 2.

TABLE 2 EC₅₀ measurements Compound EC₅₀ GLP-1 (nM) EC₅₀ GLP-2 (nM)Teduglutide 39 0.027 Liraglutide 0.029 N/A A 0.490 0.083 B 3.900 0.280 10.630 0.350 2 0.130 0.250 3 0.042 0.330 4 0.660 0.087 5 0.170 0.063 60.058 0.120 7 0.920 0.019 8 0.220 0.039 9 0.056 0.056 10 1.800 0.087 110.320 0.085 12 0.140 0.110 13 2.200 0.099 14 0.570 0.086 15 0.250 0.16016 0.073 0.680 17 0.900 0.330 18 0.190 0.210 19 0.066 0.230 20 0.5500.370 21 1.800 0.270 22 0.230 0.200 23 0.130 0.240 24 0.210 0.170 250.094 0.330 26 0.290 0.590 27 0.450 1.100 28 0.360 0.510 29 0.310 0.29030 0.310 0.380 31 0.270 0.240 32 0.380 0.460 33 0.850 0.072 34 0.2800.130 35 0.099 0.300 36 0.320 3.200 38 0.250 0.890 39 0.044 0.980 400.074 0.500 41 0.048 0.620 42 0.067 0.330 43 0.096 0.150 44 0.063 0.14045 1.400 0.360 46 0.260 0.380 47 0.440 0.048 48 0.470 0.054 49 0.2700.044 50 0.310 0.056 51 0.020 0.180 52 0.020 0.075 53 0.076 0.240 540.034 0.990 55 0.110 0.780 56 0.033 0.076 57 0.093 0.083 58 0.089 0.09059 0.088 0.110 60 0.097 0.074 61 0.130 0.200 62 0.270 0.150 63 0.3100.170 64 0.490 0.200 65 0.130 0.350 66 0.650 0.180 67 0.160 0.220 680.084 0.100 N/A = no detectable activity

Example 3: Solubility Assessment

A stock solution of the test peptide (2 mg/ml; determined from theweighed amount of peptide) in demineralized water adjusted to pH 2.5with HCl was prepared, and aliquots were diluted 1:1 in 100 mM acetatebuffer (pH 4.0 and pH 5.0), 100 mM histidine buffer (pH 6.0 and pH 7.0)and 100 mM phosphate buffer (pH 6.0, pH 7.0 and pH 7.5), respectively,and loaded in a standard flat-bottom, non-sterile 96-well UV Microplate.The absorbance of samples (single samples, n=1) at 280 and 325 nm wasmeasured in an absorbance-based plate reader, which was preheated toambient temperature (typically 25° C.). The turbidity absorbancecriterion for a peptide solubility of 1 mg/ml was an absorbance at 325nm of 0.025 absorbance units (which is 5 to 6 times the standarddeviation of 8 buffer samples in a plate). Solubility data for peptidesof the invention are shown in Table 3, below.

TABLE 3 Solubility data. Acetate Acetate Histidine Histidine PhosphatePhosphate Phosphate buffer buffer buffer buffer buffer buffer bufferCdp. pH 4 pH 5 pH 6 pH 7 pH 6 pH 7 pH 7.5 Teduglutide II II II SS II IISS 17 II II SS SS SS SS SS 18 II II SS SS SS SS SS 19 II II SS SS SS SSSS 20 II II SS SS SS SS SS 22 II II SS SS SS SS SS 23 II II SS SS SS SSSS 24 II II SS SS SS SS SS 25 II II SS SS SS SS SS 26 SS II II SS II SSSS 27 II II SS SS SS SS SS 28 II II SS SS SS SS SS *SS indicatessolubility ≥ 1 mg/ml **II indicates solubility < 1 mg/ml

Example 4: Chemical Stability

Samples of each test peptide were dissolved in MILLIQ™ water, and the pHof the solution was adjusted to pH 6, 7, 7.5 or 9 using either HCl orNaOH. The final peptide concentration was 0.2 mg/ml. Samples were placedin glass vials and incubated at 40 ° C. The samples were analyzed byRP-HPLC on a C18 column with gradient elution using anacetonitrile/TFA/water eluent system. The area-percentage (area-%) ofthe main peak after incubation time T=t (relative to time T=0) wasdetermined by UV spectroscopy at 220 nm.

The purity was first determined as follows:

Purity (area-%)=(area of main peak/total area of all peaks)×100.

The purity was then normalized between time points by setting purity attime 0 (T=0) to 100 for each pH value for a given peptide, as follows:

Normalized area-% at time t (T=t)=[area-% (T=t)/area-% (T=0)]×100.

The chemical stability assessment results after 14-day incubation (inthe form of normalized purity values) are summarized in Table 4.

TABLE 4 Chemical stability data. pH 6 pH 7 normalized normalizedCompound stability stability Teduglutide A C B B A A 18 A A 19 A A 22 AB 23 A B 24 B A 25 A 26 A 28 A 29 A B 30 A 31 A A 2 A A 5 A A 11 A 14 A53 A 42 A 55 A 58 A 67 A 68 A 32 A 33 A 34 A Key: A—>90% normalisedstability; B—>80% stability; C—<80% normalized stability.

Example 5: Effect on Fasting Glucose and Intestinal Weight in NormalMice

Normal chow-fed C57BL/6J male mice were used. The mice were kept instandard housing conditions, light-, temperature-, andhumidity-controlled room (12:12 h light-dark cycle, with lights on at06.00-18.00 h; 20-22° C.; 50-80% relative humidity). Each dosing groupconsisted of 6 animals. Mice were dosed once daily with 100 nmol/kg withthe test compounds or vehicle for 4 days via subcutaneousadministration.

On day 0 mice were fasted and blood glucose levels measured after asingle s.c. injection with peptides. Animals were sacrificed 24 hoursafter final dosing on day 3, and small intestinal wet weights weremeasured.

All test compounds (100 nmol/kg) reduced fasting blood glucose levelscompared to vehicle group (Table 5).

All test compounds (100 nmol/kg) increased small intestine wet weight ascompared to the vehicle-treated mice (Table 5).

TABLE 5 Effects on fasting blood glucose levels and small intestinalweight. Fasting blood glucose Small intestinal wet Treatment (mM) weight(g) Vehicle 8.99 0.80 Cpd. 18 5.26 1.37 Cpd. 48 5.08 1.41 Cpd. 50 5.571.43 Cpd. 5 4.60 1.35 Cpd. 8 5.28 1.27 Cpd. 9 4.98 1.11 Cpd. 52 4.691.09

1. A dual agonist represented by the formula:R¹—X*—R², wherein: R¹ is hydrogen (Hy), C₁₋₄ alkyl, acetyl, formyl,benzoyl or trifluoroacetyl; R² is NH₂ or OH; X* is a peptideH[Aib]EGSFTSELATILDψQAARDFIAWLIQHKITD (SEQ ID NO: 38); ψ is an L or DLys residue whose side chain is conjugated to a substituent of formulaZ¹—Z²— and Z¹—Z²— is [17-carboxy-heptadecanoyl]-isoGlu-, or apharmaceutically acceptable salt thereof.
 2. The dual agonist accordingto claim 1, wherein X* has the sequence: (SEQ ID NO: 85)H[Aib]EGSFTSELATILD[K*]QAARDFIAWLIQHKITD;

wherein K* indicates an L lysine residue in which the side chain isconjugated to the substituent Z¹—Z²—, or a pharmaceutically acceptablesalt thereof.
 3. The dual agonist according to claim 2, which is:(SEQ ID NO: 200) Hy-H[Aib]EGSFTSELATILD[K([17-carboxy-heptadecanoyl]-isoGlu)]QAARDFIAWLIQHKITD-OH,

or a pharmaceutically acceptable salt thereof.
 4. A pharmaceuticalcomposition comprising the dual agonist or pharmaceutically acceptablesalt thereof according to claim 1, in admixture with a pharmaceuticallyacceptable carrier, an excipient or a vehicle.
 5. A pharmaceuticalcomposition comprising the dual agonist or pharmaceutically acceptablesalt thereof according to claim 3, in admixture with a pharmaceuticallyacceptable carrier, an excipient or a vehicle.